Humanized antibodies that recognize beta amyloid peptide

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with amyloid deposits of Aβ in the brain of a patient. Preferred agents include humanized antibodies.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.10/388,389, filed Mar. 12, 2003, which is a continuation-in-part of U.S.application Ser. No. 10/010,942, filed Dec. 6, 2001, U.S. PublicationNo. 20030165496 A1, which is a an application claiming the benefit under35 U.S.C. 119(e) of U.S. Application No. 60/251,892 filed Dec. 6, 2000,each of which is incorporated herein by reference for all purposes.

[0002] U.S. application Ser. No. 10/010,942 is also acontinuation-in-part of U.S. application Ser. No. 09/580,015 filed May26, 2000, which is a continuation-in-part of U.S. application Ser. No.09/322,289, filed May 28, 1999, which is a continuation-in-part of U.S.application Ser. No. 09/201,430, filed Nov. 30, 1998, which is a anapplication claiming the benefit under 35 U.S.C. 119(e) of U.S.Application No. 60/080,970, filed Apr. 7, 1998, each of which isincorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

[0003] Alzheimer's disease (AD) is a progressive disease resulting insenile dementia. See generally Selkoe, TINS 16:403 (1993); Hardy et al.,WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53:438 (1994); Duff etal., Nature 373:476 (1995); Games et al., Nature 373:523 (1995). Broadlyspeaking, the disease falls into two categories: late onset, whichoccurs in old age (65+years) and early onset, which develops well beforethe senile period, i.e., between 35 and 60 years. In both types ofdisease, the pathology is the same but the abnormalities tend to be moresevere and widespread in cases beginning at an earlier age. The diseaseis characterized by at least two types of lesions in the brain,neurofibrillary tangles and senile plaques. Neurofibrillary tangles areintracellular deposits of microtubule associated tau protein consistingof two filaments twisted about each other in pairs. Senile plaques(i.e., amyloid plaques) are areas of disorganized neuropil up to 150 μmacross with extracellular amyloid deposits at the center which arevisible by microscopic analysis of sections of brain tissue. Theaccumulation of amyloid plaques within the brain is also associated withDown's syndrome and other cognitive disorders.

[0004] The principal constituent of the plaques is a peptide termed Aβor β-amyloid peptide. Aβ peptide is a 4-kDa internal fragment of 39-43amino acids of a larger transmembrane glycoprotein named protein termedamyloid precursor protein (APP). As a result of proteolytic processingof APP by different secretase enzymes, Aβ is primarily found in both ashort form, 40 amino acids in length, and a long form, ranging from42-43 amino acids in length. Part of the hydrophobic transmembranedomain of APP is found at the carboxy end of Aβ, and may account for theability of Aβ to aggregate into plaques, particularly in the case of thelong form. Accumulation of amyloid plaques in the brain eventually leadsto neuronal cell death. The physical symptoms associated with this typeof neural deterioration characterize Alzheimer's disease.

[0005] Several mutations within the APP protein have been correlatedwith the presence of Alzheimer's disease. See, e.g., Goate et al.,Nature 349:704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al.Nature 353:844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science254:97 (1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet.1:345 (1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20: 154 (1997)).

[0006] Mouse models have been used successfully to determine thesignificance of amyloid plaques in Alzheimer's (Games et al., supra,Johnson-Wood et al., Proc. Natl. Acad. Sci. USA 94:1550 (1997)). Inparticular, when PDAPP transgenic mice, (which express a mutant form ofhuman APP and develop Alzheimer's disease at a young age), are injectedwith the long form of Aβ, they display both a decrease in theprogression of Alzheimer's and an increase in antibody titers to the Aβpeptide (Schenk et al., Nature 400, 173 (1999)). The observationsdiscussed above indicate that Aβ, particularly in its long form, is acausative element in Alzheimer's disease.

[0007] McMichael, EP 526,511, proposes administration of homeopathicdosages (less than or equal to 10⁻² mg/day) of Aβ to patients withpreestablished AD. In a typical human with about 5 liters of plasma,even the upper limit of this dosage would be expected to generate aconcentration of no more than 2 pg/ml. The normal concentration of Aβ inhuman plasma is typically in the range of 50-200 pg/ml (Seubert et al.,Nature 359:325 (1992)). Because EP 526,511 's proposed dosage wouldbarely alter the level of endogenous circulating Aβ and because EP526,511 does not recommend use of an adjuvant, as an immunostimulant, itseems implausible that any therapeutic benefit would result.

[0008] Accordingly, there exists the need for new therapies and reagentsfor the treatment of Alzheimer's disease, in particular, therapies andreagents capable of effecting a therapeutic benefit at physiologic(e.g., non-toxic) doses.

SUMMARY OF THE INVENTION

[0009] The present invention features new immunological reagents, inparticular, therapeutic antibody reagents for the prevention andtreatment of amyloidogenic disease (e.g., Alzheimer's disease). Theinvention is based, at least in part, on the identification andcharacterization of two monoclonal antibodies that specifically bind toAβ peptide and are effective at reducing plaque burden and/or reducingthe neuritic dystrophy associated with amyloidogenic disorders.Structural and functional analysis of these antibodies leads to thedesign of various humanized antibodies for prophylactic and/ortherapeutic use. In particular, the invention features humanization ofthe variable regions of these antibodies and, accordingly provides forhumanized immunoglobulin or antibody chains, intact humanizedimmunoglobulins or antibodies, and functional immunoglobulin or antibodyfragments, in particular, antigen binding fragments, of the featuredantibodies.

[0010] Polypeptides comprising the complementarity determining regionsof the featured monoclonal antibodies are also disclosed, as arepolynucleotide reagents, vectors and host suitable for encoding saidpolypeptides.

[0011] Methods of treatment of amyloidogenic diseases or disorders(e.g., Alzheimer's disease) are disclosed, as are pharmaceuticalcompositions and kits for use in such applications.

[0012] Also featured are methods of identifying residues within thefeatured monoclonal antibodies which are important for properimmunologic function and for identifying residues which are amenable tosubstitution in the design of humanized antibodies having improvedbinding affinities and/or reduced immunogenicity, when used astherapeutic reagents.

[0013] Also featured are antibodies (e.g., humanized antibodies) havingaltered effector functions, and therapeutic uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 depicts an alignment of the amino acid sequences of thelight chain of mouse 3D6, humanized 3D6, Kabat ID 109230 and germlineA19 antibodies. CDR regions are indicated by arrows. Bold italicsindicate rare murine residues. Bold indicates packing (VH+VL) residues.Solid fill indicates canonical/CDR interacting residues. Asterisksindicate residues selected for backmutation in humanized 3D6, version 1.

[0015]FIG. 2 depicts an alignment of the amino acid sequences of theheavy chain of mouse 3D6, humanized 3D6, Kabat ID 045919 and germlineVH3-23 antibodies. Annotation is the same as for FIG. 1.

[0016]FIG. 3 graphically depicts the Aβ binding properties of 3D6,chimeric 3D6 and 10D5. FIG. 3A is a graph depicting binding of Aβ tochimeric 3D6 (PK1614) as compared to murine 3D6. FIG. 3B is a graphdepicting competition of biotinylated 3D6 versus unlabeled 3D6, PK1614and 10D5 for binding to Aβ.

[0017]FIG. 4 depicts a homology model of 3D6 VH and VL, showing α-carbonbackbone trace. VH is shown in as a stippled line, and VL is shown as asolid line. CDR regions are indicated in ribbon form.

[0018]FIG. 5 graphically depicts the Aβ binding properties of chimeric3D6 and humanized 3D6. FIG. 5A depicts ELISA results measuring thebinding of humanized 3D6v1 and chimeric 3D6 to aggregated Aβ. FIG. 5Bdepicts ELISA results measuring the binding of humanized 3D6v1 andhumanized 3D6v2 to aggregated Aβ.

[0019]FIG. 6 is a graph quantitating the binding of humanized 3D6 andchimeric 3D6 to Aβ plaques from brain sections of PDAPP mice.

[0020]FIG. 7 is a graph showing results of a competitive binding assaytesting the ability of humanized 3D6 versions 1 and 2, chimeric 3D6,murine 3D6, and 10D5 to compete with murine 3D6 for binding to Aβ.

[0021]FIG. 8 graphically depicts of an ex vivo phagocytosis assaytesting the ability of humanized 3D6v2, chimeric 3D6, and human IgG tomediate the uptake of Aα by microglial cells.

[0022]FIG. 9 depicts an alignment of the 10D5 VL and 3D6 VL amino acidsequences. Bold indicates residues that match 10D5 exactly.

[0023]FIG. 10 depicts an alignment of the 10D5 VH and 3D6 VH amino acidsequences. Bold indicates residues that match 10D5 exactly.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention features new immunological reagents andmethods for preventing or treating Alzheimer's disease or otheramyloidogenic diseases. The invention is based, at least in part, on thecharacterization of two monoclonal immunoglobulins, 3D6 and 10D5,effective at binding beta amyloid protein (Aβ) (e.g., binding solubleand/or aggregated Aβ), mediating phagocytosis (e.g., of aggregated Aβ),reducing plaque burden and/or reducing neuritic dystrophy (e.g., inpatient). The invention is further based on the determination andstructural characterization of the primary and secondary structure ofthe variable light and heavy chains of these immunoglobulins and theidentification of residues important for activity and immunogenicity.

[0025] Immunoglobulins are featured which include a variable lightand/or variable heavy chain of the preferred monoclonal immunoglobulinsdescribed herein. Preferred immunoglobulins, e.g., therapeuticimmunoglobulins, are featured which include a humanized variable lightand/or humanized variable heavy chain. Preferred variable light and/orvariable heavy chains include a complementarity determining region (CDR)from the monoclonal immunoglobulin (e.g., donor immunoglobulin) andvariable framework regions substantially from a human acceptorimmunoglobulin. The phrase “substantially from a human acceptorimmunoglobulin” means that the majority or key framework residues arefrom the human acceptor sequence, allowing however, for substitution ofresidues at certain positions with residues selected to improve activityof the humanized immunoglobulin (e.g., alter activity such that it moreclosely mimics the activity of the donor immunoglobulin) or selected todecrease the immunogenicity of the humanized immunoglobulin.

[0026] In one embodiment, the invention features a humanizedimmunoglobulin light or heavy chain that includes 3D6 variable regioncomplementarity determining regions (CDRs) (i.e., includes one, two orthree CDRs from the light chain variable region sequence set forth asSEQ ID NO:2 or includes one, two or three CDRs from the heavy chainvariable region sequence set forth as SEQ ID NO:4), and includes avariable framework region substantially from a human acceptorimmunoglobulin light or heavy chain sequence, provided that at least oneresidue of the framework residue is backmutated to a correspondingmurine residue, wherein said backmutation does not substantially affectthe ability of the chain to direct Aβ binding.

[0027] In another embodiment, the invention features a humanizedimmunoglobulin light or heavy chain that includes 3D6 variable regioncomplementarity determining regions (CDRs) (e.g., includes one, two orthree CDRs from the light chain variable region sequence set forth asSEQ ID NO:2 and/or includes one, two or three CDRs from the heavy chainvariable region sequence set forth as SEQ ID NO:4), and includes avariable framework region substantially from a human acceptorimmunoglobulin light or heavy chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 3D6 light or heavy chain variable regionsequence, where the framework residue is selected from the groupconsisting of (a) a residue that non-covalently binds antigen directly;(b) a residue adjacent to a CDR; (c) a CDR-interacting residue (e.g.,identified by modeling the light or heavy chain on the solved structureof a homologous known immunoglobulin chain); and (d) a residueparticipating in the VL-VH interface.

[0028] In another embodiment, the invention features a humanizedimmunoglobulin light or heavy chain that includes 3D6 variable regionCDRs and variable framework regions from a human acceptor immunoglobulinlight or heavy chain sequence, provided that at least one frameworkresidue is substituted with the corresponding amino acid residue fromthe mouse 3D6 light or heavy chain variable region sequence, where theframework residue is a residue capable of affecting light chain variableregion conformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, anunusual residue, or a glycoslyation site residue on the surface of thestructural model.

[0029] In another embodiment, the invention features a humanizedimmunoglobulin light chain that includes 3D6 variable region CDRs (e.g.,from the 3D6 light chain variable region sequence set forth as SEQ IDNO:2), and includes a human acceptor immunoglobulin variable frameworkregion, provided that at least one framework residue selected from thegroup consisting of L1, L2, L36 and L46 (Kabat numbering convention) issubstituted with the corresponding amino acid residue from the mouse 3D6light chain variable region sequence. In another embodiment, theinvention features a humanized immunoglobulin heavy chain that includes3D6 variable region CDRs (e.g., from the 3D6 heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a human acceptorimmunoglobulin variable framework region, provided that at least oneframework residue selected from the group consisting of H49, H93 and H94(Kabat numbering convention) is substituted with the corresponding aminoacid residue from the mouse 3D6 heavy chain variable region sequence.

[0030] Preferred light chains include kappa II framework regions of thesubtype kappa II (Kabat convention), for example, framework regions fromthe acceptor immunoglobulin Kabat ID 019230, Kabat ID 005131, Kabat ID005058, Kabat ID 005057, Kabat ID 005059, Kabat ID U21040 and Kabat IDU41645. Preferred heavy chains include framework regions of the subtypeIII (Kabat convention), for example, framework regions from the acceptorimmunoglobulin Kabat ID 045919, Kabat ID 000459, Kabat ID 000553, KabatID 000386 and Kabat ID M23691.

[0031] In one embodiment, the invention features a humanizedimmunoglobulin light or heavy chain that includes 10D5 variable regioncomplementarity determining regions (CDRs) (i.e., includes one, two orthree CDRs from the light chain variable region sequence set forth asSEQ ID NO:14 or includes one, two or three CDRs from the heavy chainvariable region sequence set forth as SEQ ID NO:16), and includes avariable framework region substantially from a human acceptorimmunoglobulin light or heavy chain sequence, provided that at least oneresidue of the framework residue is backmutated to a correspondingmurine residue, wherein said backmutation does not substantially affectthe ability of the chain to direct Aβ binding.

[0032] In another embodiment, the invention features a humanizedimmunoglobulin light or heavy chain that includes 10D5 variable regioncomplementarity determining regions (CDRs) (e.g., includes one, two orthree CDRs from the light chain variable region sequence set forth asSEQ ID NO:14 and/or includes one, two or three CDRs from the heavy chainvariable region sequence set forth as SEQ ID NO:16), and includes avariable framework region substantially from a human acceptorimmunoglobulin light or heavy chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 3D6 light or heavy chain variable regionsequence, where the framework residue is selected from the groupconsisting of (a) a residue that non-covalently binds antigen directly;(b) a residue adjacent to a CDR; (c) a CDR-interacting residue (e.g.,identified by modeling the light or heavy chain on the solved structureof a homologous known immunoglobulin chain); and (d) a residueparticipating in the VL-VH interface.

[0033] In another embodiment, the invention features a humanizedimmunoglobulin light or heavy chain that includes 10D5 variable regionCDRs and variable framework regions from a human acceptor immunoglobulinlight or heavy chain sequence, provided that at least one frameworkresidue is substituted with the corresponding amino acid residue fromthe mouse 10D5 light or heavy chain variable region sequence, where theframework residue is a residue capable of affecting light chain variableregion conformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, anunusual residue, or a glycoslyation site residue on the surface of thestructural model.

[0034] In another embodiment, the invention features, in addition to thesubstitutions described above, a substitution of at least one rare humanframework residue. For example, a rare residue can be substituted withan amino acid residue which is common for human variable chain sequencesat that position. Alternatively, a rare residue can be substituted witha corresponding amino acid residue from a homologous germline variablechain sequence (e.g., a rare light chain framework residue can besubstituted with a corresponding germline residue from an A1, A17, A18,A2, or A19 germline immunoglobulin sequence or a rare heavy chainframework residue can be substituted with a corresponding germlineresidue from a VH3-48, VH3-23, VH3-7, VH3-21 or VH3-11 germlineimmunoglobulin sequence.

[0035] In another embodiment, the invention features a humanizedimmunoglobulin that includes a light chain and a heavy chain, asdescribed above, or an antigen-binding fragment of said immunoglobulin.In an exemplary embodiment, the humanized immunoglobulin binds (e.g.,specifically binds) to beta amyloid peptide (Aβ) with a binding affinityof at least 10⁷ M⁻¹, 10⁸ M⁻¹, or 10⁹ M⁻¹. In another embodiment, theimmunoglobulin or antigen binding fragment includes a heavy chain havingisotype γ1. In another embodiment, the immunoglobulin or antigen bindingfragment binds (e.g., specifically binds) to both soluble beta amyloidpeptide (Aβ) and aggregated Aβ. In another embodiment, theimmunoglobulin or antigen binding fragment mediates phagocytosis (e.g.,induces phagocytosis) of beta amyloid peptide (Aβ). In yet anotherembodiment, the immunoglobulin or antigen binding fragment crosses theblood-brain barrier in a subject. In yet another embodiment, theimmunoglobulin or antigen binding fragment reduces both beta amyloidpeptide (Aβ) burden and neuritic dystrophy in a subject.

[0036] In another embodiment, the invention features chimericimmunoglobulins that include 3D6 variable regions (e.g., the variableregion sequences set forth as SEQ ID NO:2 or SEQ ID NO:4). As usedherein, an antibody or immunoglobulin sequence comprising a VL and/or VHsequence as set forth in, for example, SEQ ID NO:2 or SEQ ID NO:4 cancomprise either the full VL or VH sequence or can comprise the mature VLor VH sequence (i.e., mature peptide without the signal or leaderpeptide). In yet another embodiment, the invention features animmunoglobulin, or antigen-binding fragment thereof, including avariable heavy chain region as set forth in SEQ ID NO:8 and a variablelight chain region as set forth in SEQ ID NO:5. In yet anotherembodiment, the invention features an immunoglobulin, or antigen-bindingfragment thereof, including a variable heavy chain region as set forthin SEQ ID NO:12 and a variable light chain region as set forth in SEQ IDNO:11. In another embodiment, the invention features chimericimmunoglobulins that include 10D5 variable regions (e.g., the variableregion sequences set forth as SEQ ID NO:14 or SEQ ID NO:16). In yetanother embodiment, the immunoglobulin, or antigen-binding fragmentthereof, further includes constant regions from IgG1.

[0037] The immunoglobulins described herein are particularly suited foruse in therapeutic methods aimed at preventing or treating amyloidogenicdiseases. In one embodiment, the invention features a method ofpreventing or treating an amyloidogenic disease (e.g., Alzheimer'sdisease) that involves administering to the patient an effective dosageof a humanized immunoglobulin as described herein. In anotherembodiment, the invention features pharmaceutical compositions thatinclude a humanized immunoglobulin as described herein and apharmaceutical carrier. Also featured are isolated nucleic acidmolecules, vectors and host cells for producing the immunoglobulins orimmunoglobulin fragments or chains described herein, as well as methodsfor producing said immunoglobulins, immunoglobulin fragments orimmunoglobulin chains The present invention further features a methodfor identifying 3D6 or 10D5 residues amenable to substitution whenproducing a humanized 3D6 or 10D5 immunoglobulin, respectively. Forexample, a method for identifying variable framework region residuesamenable to substitution involves modeling the three-dimensionalstructure of the 3D6 or 10D5 variable region on a solved homologousimmunoglobulin structure and analyzing said model for residues capableof affecting 3D6 or 10D5 immunoglobulin variable region conformation orfunction, such that residues amenable to substitution are identified.The invention further features use of the variable region sequence setforth as SEQ ID NO:2 or SEQ ID NO:4, or any portion thereof, inproducing a three-dimensional image of a 3D6 immunoglobulin, 3D6immunoglobulin chain, or domain thereof. Also featured is the use of thevariable region sequence set forth as SEQ ID NO:14 or SEQ ID NO:16, orany portion thereof, in producing a three-dimensional image of a 10D5immunoglobulin, 10D5 immunoglobulin chain, or domain thereof.

[0038] The present invention further features immunoglobulins havingaltered effector function, such as the ability to bind effectormolecules, for example, complement or a receptor on an effector cell. Inparticular, the immunoglobulin of the invention has an altered constantregion, e.g., Fc region, wherein at least one amino acid residue in theFc region has been replaced with a different residue or side chain. Inone embodiment, the modified immunoglobulin is of the IgG class,comprises at least one amino acid residue replacement in the Fc regionsuch that the immunoglobulin has an altered effector function, e.g., ascompared with an unmodified immunoglobulin. In particular embodiments,the immunoglobulin of the invention has an altered effector functionsuch that it is less immunogenic (e.g., does not provoke undesiredeffector cell activity, lysis, or complement binding), has improvedamyloid clearance properties, and/or has a desirable half-life.

[0039] Prior to describing the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

[0040] The term “immunoglobulin” or “antibody” (used interchangeablyherein) refers to an antigen-binding protein having a basicfour-polypeptide chain structure consisting of two heavy and two lightchains, said chains being stabilized, for example, by interchaindisulfide bonds, which has the ability to specifically bind antigen.Both heavy and light chains are folded into domains. The term “domain”refers to a globular region of a heavy or light chain polypeptidecomprising peptide loops (e.g., comprising 3 to 4 peptide loops)stabilized, for example, by β-pleated sheet and/or intrachain disulfidebond. Domains are further referred to herein as “constant” or“variable”based on the relative lack of sequence variation within thedomains of various class members in the case of a “constant” domain, orthe significant variation within the domains of various class members inthe case of a “variable” domain. “Constant” domains on the light chainare referred to interchangeably as “light chain constant regions”,“light chain constant domains”, “CL” regions or “CL” domains).“Constant” domains on the heavy chain are referred to interchangeably as“heavy chain constant regions”, “heavy chain constant domains”, “CH”regions or “CH” domains). “Variable” domains on the light chain arereferred to interchangeably as “light chain variable regions”, “lightchain variable domains”, “VL” regions or “VL” domains). “Variable”domains on the heavy chain are referred to interchangeably as “heavychain constant regions”, “heavy chain constant domains”, “CH” regions or“CH” domains).

[0041] The term “region” refers to a part or portion of an antibodychain and includes constant or variable domains as defined herein, aswell as more discrete parts or portions of said domains. For example,light chain variable domains or regions include “complementaritydetermining regions” or “CDRs” interspersed among “framework regions” or“FRs”, as defined herein.

[0042] Immunoglobulins or antibodies can exist in monomeric or polymericform. The term “antigen-binding fragment” refers to a polypeptidefragment of an immunoglobulin or antibody binds antigen or competes withintact antibody (i.e., with the intact antibody from which they werederived) for antigen binding (i.e., specific binding). The term“conformation” refers to the tertiary structure of a protein orpolypeptide (e.g., an antibody, antibody chain, domain or regionthereof). For example, the phrase “light (or heavy) chain conformation”refers to the tertiary structure of a light (or heavy) chain variableregion, and the phrase “antibody conformation” or “antibody fragmentconformation” refers to the tertiary structure of an antibody orfragment thereof.

[0043] “Specific binding” of an antibody mean that the antibody exhibitsappreciable affinity for antigen or a preferred epitope and, preferably,does not exhibit significant crossreactivity. “Appreciable” or preferredbinding include binding with an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁷ M⁻¹, preferably greaterthan 10⁸ M⁻¹ are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities, for example, 10⁶ to 10¹⁰ M⁻¹, preferably 10⁷ to 10¹⁰ M⁻¹,more preferably 10⁸ to 10¹⁰ M⁻¹. An antibody that “does not exhibitsignificant crossreactivity” is one that will not appreciably bind to anundesirable entity (e.g., an undesirable proteinaceous entity). Forexample, an antibody that specifically binds to Aβ will appreciably bindAβ but will not significantly react with non-Aβ proteins or peptides(e.g., non-Aβ proteins or peptides included in plaques). An antibodyspecific for a preferred epitope will, for example, not significantlycrossreact with remote epitopes on the same protein or peptide. Specificbinding can be determined according to any art-recognized means fordetermining such binding. Preferably, specific binding is determinedaccording to Scatchard analysis and/or competitive binding assays.

[0044] Binding fragments are produced by recombinant DNA techniques, orby enzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

[0045] The term “humanized immunoglobulin” or “humanized antibody”refers to an immunoglobulin or antibody that includes at least onehumanized immunoglobulin or antibody chain (i.e., at least one humanizedlight or heavy chain). The term “humanized immunoglobulin chain” or“humanized antibody chain” (i.e., a “humanized immunoglobulin lightchain” or “humanized immunoglobulin heavy chain”) refers to animmunoglobulin or antibody chain (i.e., a light or heavy chain,respectively) having a variable region that includes a variableframework region substantially from a human immunoglobulin or antibodyand complementarity determining regions (CDRs) (e.g., at least one CDR,preferably two CDRs, more preferably three CDRs) substantially from anon-human immunoglobulin or antibody, and further includes constantregions (e.g., at least one constant region or portion thereof, in thecase of a light chain, and preferably three constant regions in the caseof a heavy chain). The term “humanized variable region” (e.g.,“humanized light chain variable region” or “humanized heavy chainvariable region”) refers to a variable region that includes a variableframework region substantially from a human immunoglobulin or antibodyand complementarity determining regions (CDRs) substantially from anon-human immunoglobulin or antibody.

[0046] The phrase “substantially from a human immunoglobulin orantibody” or “substantially human” means that, when aligned to a humanimmunoglobulin or antibody amino sequence for comparison purposes, theregion shares at least 80-90%, preferably 90-95%, more preferably 95-99%identity (i.e., local sequence identity) with the human framework orconstant region sequence, allowing, for example, for conservativesubstitutions, consensus sequence substitutions, germline substitutions,backmutations, and the like. The introduction of conservativesubstitutions, consensus sequence substitutions, germline substitutions,backmutations, and the like, is often referred to as “optimization” of ahumanized antibody or chain. The phrase “substantially from a non-humanimmunoglobulin or antibody” or “substantially non-human” means having animmunoglobulin or antibody sequence at least 80-95%, preferably 90-95%,more preferably, 96%, 97%, 98%, or 99% identical to that of a non-humanorganism, e.g., a non-human mammal.

[0047] Accordingly, all regions or residues of a humanizedimmunoglobulin or antibody, or of a humanized immunoglobulin or antibodychain, except possibly the CDRs, are substantially identical to thecorresponding regions or residues of one or more native humanimmunoglobulin sequences. The term “corresponding region” or“corresponding residue” refers to a region or residue on a second aminoacid or nucleotide sequence which occupies the same (i.e., equivalent)position as a region or residue on a first amino acid or nucleotidesequence, when the first and second sequences are optimally aligned forcomparison purposes.

[0048] The terms “humanized immunoglobulin” or “humanized antibody” arenot intended to encompass chimeric immunoglobulins or antibodies, asdefined infra. Although humanized immunoglobulins or antibodies arechimeric in their construction (i.e., comprise regions from more thanone species of protein), they include additional features (i.e.,variable regions comprising donor CDR residues and acceptor frameworkresidues) not found in chimeric immunoglobulins or antibodies, asdefined herein.

[0049] The term “significant identity” means that two polypeptidesequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 50-60% sequenceidentity, preferably 60-70% sequence identity, more preferably 70-80%sequence identity, more preferably at least 80-90% identity, even morepreferably at least 90-95% identity, and even more preferably at least95% sequence identity or more (e.g., 99% sequence identity or more). Theterm “substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80-90% sequence identity, preferably 90-95%sequence identity, and more preferably at least 95% sequence identity ormore (e.g., 99% sequence identity or more). For sequence comparison,typically one sequence acts as a reference sequence, to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. The terms “sequence identity” and “sequence identity” areused interchangeably herein.

[0050] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., Current Protocols in Molecular Biology). Oneexample of algorithm that is suitable for determining percent sequenceidentity and sequence similarity is the BLAST algorithm, which isdescribed in Altschul et al., J. Mol. Biol. 215:403 (1990). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (publicly accessible through theNational Institutes of Health NCBI internet server). Typically, defaultprogram parameters can be used to perform the sequence comparison,although customized parameters can also be used. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3, anexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0051] Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gln, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

[0052] Preferably, humanized immunoglobulins or antibodies bind antigenwith an affinity that is within a factor of three, four, or five of thatof the corresponding non-human antibody. For example, if the nonhumanantibody has a binding affinity of 10⁹ M⁻¹, humanized antibodies willhave a binding affinity of at least 3×10⁹ M^(−l), 4×10⁹ M⁻¹ or 10⁹ M⁻¹.When describing the binding properties of an immunoglobulin or antibodychain, the chain can be described based on its ability to “directantigen (e.g., Aβ) binding”. A chain is said to “direct antigen binding”when it confers upon an intact immunoglobulin or antibody (or antigenbinding fragment thereof) a specific binding property or bindingaffinity. A mutation (e.g., a backmutation) is said to substantiallyaffect the ability of a heavy or light chain to direct antigen bindingif it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

[0053] The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

[0054] An “antigen” is an entity (e.g., a protenaceous entity orpeptide) to which an antibody specifically binds.

[0055] The term “epitope” or “antigenic determinant” refers to a site onan antigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

[0056] Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen, i.e., a competitivebinding assay. Competitive binding is determined in an assay in whichthe immunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

[0057] An epitope is also recognized by immunologic cells, for example,B cells and/or T cells. Cellular recognition of an epitope can bedetermined by in vitro assays that measure antigen-dependentproliferation, as determined by ³H-thymidine incorporation, by cytokinesecretion, by antibody secretion, or by antigen-dependent killing(cytotoxic T lymphocyte assay).

[0058] Exemplary epitopes or antigenic determinants can be found withinthe human amyloid precursor protein (APP), but are preferably foundwithin the Aβ peptide of APP. Multiple isoforms of APP exist, forexample APP⁶⁹⁵ APP⁷⁵¹ and APP⁷⁷⁰. Amino acids within APP are assignednumbers according to the sequence of the APP⁷⁷⁰ isoform (see e.g.,GenBank Accession No. P05067, also set forth as SEQ ID NO:38). Aβ (alsoreferred to herein as beta amyloid peptide and A-beta) peptide is a˜4-kDa internal fragment of 39-43 amino acids of APP (Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43). Aβ40, for example, consists of residues 672-711 of APPand Aβ42 consists of residues 673-713 of APP. As a result of proteolyticprocessing of APP by different secretase enzymes iv vivo or in situ, Aβis found in both a “short form”, 40 amino acids in length, and a “longform”, ranging from 42-43 amino acids in length. Preferred epitopes orantigenic determinants, as described herein, are located within theN-terminus of the Aβ peptide and include residues within amino acids1-10 of Aβ, preferably from residues 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7 ofAβ42. Additional referred epitopes or antigenic determinants includeresidues 2-4, 5, 6, 7 or 8 of Aβ, residues 3-5, 6, 7, 8 or 9 of Aβ, orresidues 4-7, 8, 9 or 10 of Aβ42.

[0059] The term “amyloidogenic disease” includes any disease associatedwith (or caused by) the formation or deposition of insoluble amyloidfibrils. Exemplary amyloidogenic diseases include, but are not limitedto systemic amyloidosis, Alzheimer's disease, mature onset diabetes,Parkinson's disease, Huntington's disease, fronto-temporal dementia, andthe prion-related transmissible spongiform encephalopathies (kuru andCreutzfeldt-Jacob disease in humans and scrapie and BSE in sheep andcattle, respectively). Different amyloidogenic diseases are defined orcharacterized by the nature of the polypeptide component of the fibrilsdeposited. For example, in subjects or patients having Alzheimer'sdisease, β-amyloid protein (e.g., wild-type, variant, or truncatedβ-amyloid protein) is the characterizing polypeptide component of theamyloid deposit. Accordingly, Alzheimer's disease is an example of a“disease characterized by deposits of Aβ” or a “disease associated withdeposits of Aβ”, e.g. in the brain of a subject or patient. The terms“β-amyloid protein”, “β-amyloid peptide”, “β-amyloid”, “Aβ” and “Aβpeptide” are used interchangeably herein.

[0060] The term “effective dose” or “effective dosage” is defined as anamount sufficient to achieve or at least partially achieve the desiredeffect. The term “therapeutically effective dose” is defined as anamount sufficient to cure or at least partially arrest the disease andits complications in a patient already suffering from the disease.Amounts effective for this use will depend upon the severity of theinfection and the general state of the patient's own immune system.

[0061] The term “patient” includes human and other mammalian subjectsthat receive either prophylactic or therapeutic treatment.

[0062] “Soluble” or “dissociated” Aβ refers to non-aggregating ordisaggregated Aβ polypeptide. “Insoluble” Aβ refers to aggregating Aβpolypeptide, for example, Aβ held together by noncovalent bonds. Aβ(e.g., Aβ42) is believed to aggregate, at least in part, due to thepresence of hydrophobic residues at the C-terminus of the peptide (partof the transmembrane domain of APP). One method to prepare soluble Aβ isto dissolve lyophilized peptide in neat DMSO with sonication. Theresulting solution is centrifuged to remove any insoluble particulates.

[0063] The term “effector function” refers to an activity that residesin the Fc region of an antibody (e.g., an IgG antibody) and includes,for example, the ability of the antibody to bind effector molecules suchas complement and/or Fc receptors, which can control several immunefunctions of the antibody such as effector cell activity, lysis,complement-mediated activity, antibody clearance, and antibodyhalf-life.

[0064] The term “effector molecule” refers to a molecule that is capableof binding to the Fc region of an antibody (e.g., an IgG antibody)including, but not limited to, a complement protein or a Fc receptor.

[0065] The term “effector cell” refers to a cell capable of binding tothe Fc portion of an antibody (e.g., an IgG antibody) typically via anFc receptor expressed on the surface of the effector cell including, butnot limited to, lymphocytes, e.g. antigen presenting cells and T cells.

[0066] The term “Fc region” refers to a C-terminal region of an IgGantibody, in particular, the C-terminal region of the heavy chain(s) ofsaid IgG antibody. Although the boundaries of the Fc region of an IgGheavy chain can vary slightly, a Fc region is typically defined asspanning from about amino acid residue Cys226 to the carboxyl-terminusof an IGg heavy chain(s).

[0067] The term “Kabat numbering” unless otherwise stated, is defined asthe numbering of the residues in, e.g., an IgG heavy chain antibodyusing the EU index as in Kabat et al. (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)), expressly incorporatedherein by reference.

[0068] The term “Fc receptor” or “FcR” refers to a receptor that bindsto the Fc region of an antibody. Typical Fc receptors which bind to anFc region of an antibody (e.g., an IgG antibody) include, but are notlimited to, receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. Fc receptors are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

[0069] I. Immunological and Therapeutic Reagents

[0070] Immunological and therapeutic reagents of the invention compriseor consist of immunogens or antibodies, or functional or antigen bindingfragments thereof, as defined herein. The basic antibody structural unitis known to comprise a tetramer of subunits. Each tetramer is composedof two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function.

[0071] Light chains are classified as either kappa or lambda and areabout 230 residues in length. Heavy chains are classified as gamma (γ),mu (μ), alpha (α), delta (δ), or epsilon (ε), are about 450-600 residuesin length, and define the antibody's isotype as IgG, IgM, IgA, IgD andIgE, respectively. Both heavy and light chains are folded into domains.The term “domain” refers to a globular region of a protein, for example,an immunoglobulin or antibody. Immunoglobulin or antibody domainsinclude, for example, 3 or four peptide loops stabilized by β-pleatedsheet and an interchain disulfide bond. Intact light chains have, forexample, two domains (V_(L) and C_(L)) and intact heavy chains have, forexample, four or five domains (V_(H), C_(H)1, C_(H)2, and C_(H)3).

[0072] Within light and heavy chains, the variable and constant regionsare joined by a “J” region of about 12 or more amino acids, with theheavy chain also including a “D” region of about 10 more amino acids.(See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989), Ch. 7, incorporated by reference in its entirety forall purposes).

[0073] The variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. Naturally-occurring chains or recombinantly produced chains canbe expressed with a leader sequence which is removed during cellularprocessing to produce a mature chain. Mature chains can also berecombinantly produced having a non-naturally occurring leader sequence,for example, to enhance secretion or alter the processing of aparticular chain of interest.

[0074] The CDRs of the two mature chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. “FR4” also is referredto in the art as the D/J region of the variable heavy chain and the Jregion of the variable light chain. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991). An alternative structural definition hasbeen proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature342:878 (1989); and J. Mol. Biol. 186:651 (1989) (hereinaftercollectively referred to as “Chothia et al.” and incorporated byreference in their entirety for all purposes).

[0075] A. Aβ Antibodies

[0076] Therapeutic agents of the invention include antibodies thatspecifically bind to Aβ or other component of amyloid plaques. Suchantibodies can be monoclonal or polyclonal. Some such antibodies bindspecifically to the aggregated form of Aβ without binding to the solubleform. Some bind specifically to the soluble form without binding to theaggregated form. Some bind to both aggregated and soluble forms. Somesuch antibodies bind to a naturally occurring short form of Aβ (i.e.,Aβ39, 40 or 41) without binding to a naturally occurring long form of Aβ(i.e., Aβ42 and Aβ43). Some antibodies bind to a long form of Aβ withoutbinding to a short form. Some antibodies bind to Aβ without binding tofull-length amyloid precursor protein. Antibodies used in therapeuticmethods preferably have an intact constant region or at least sufficientof the constant region to interact with an Fc receptor. Human isotypeIgG1 is preferred because of it having highest affinity of humanisotypes for the FcRI receptor on phagocytic cells. Bispecific Fabfragments can also be used, in which one arm of the antibody hasspecificity for Aβ, and the other for an Fc receptor. Preferredantibodies bind to Aβ with a binding affinity greater than (or equal to)about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹ (including affinities intermediateof these values).

[0077] Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of Aβ. However, polyclonalsera can be specific to a particular segment of Aβ, such as Aβ1-10.Monoclonal antibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Prophylactic andtherapeutic efficacy of antibodies can be tested using the transgenicanimal model procedures described in the Examples. Preferred monoclonalantibodies bind to an epitope within residues 1-10 of Aβ (with the firstN terminal residue of natural Aβ designated 1). Some preferredmonoclonal antibodies bind to an epitope within amino acids 1-5, andsome to an epitope within 5-10. Some preferred antibodies bind toepitopes within amino acids 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7. Somepreferred antibodies bind to an epitope starting at resides 1-3 andending at residues 7-11 of Aβ. Less preferred antibodies include thosebinding to epitopes with residues 10-15, 15-20, 25-30, 10-20, 20, 30, or10-25 of Aβ. It is recommended that such antibodies be screened foractivity in the mouse models described in the Examples before use. Forexample, it has been found that certain antibodies to epitopes withinresidues 10-18, 16-24, 18-21 and 33-42 lack activity (e.g., lack theability to reduce plaque burden and/or resolve the neuritic pathologyassociated with Alzheimer's disease). In some methods, multiplemonoclonal antibodies having binding specificities to different epitopesare used. Such antibodies can be administered sequentially orsimultaneously. Antibodies to amyloid components other than Aβ can alsobe used (e.g., administered or co-administered). For example, antibodiescan be directed to the amyloid associated protein synuclein.

[0078] When an antibody is said to bind to an epitope within specifiedresidues, such as Aβ 1-5 for example, what is meant is that the antibodyspecifically binds to a polypeptide containing the specified residues(i.e., Aβ 1-5 in this an example). Such an antibody does not necessarilycontact every residue within Aβ 1-5. Nor does every single amino acidsubstitution or deletion with in Aβ1-5 necessarily significantly affectbinding affinity. Epitope specificity of an antibody can be determined,for example, by forming a phage display library in which differentmembers display different subsequences of Aβ. The phage display libraryis then selected for members specifically binding to an antibody undertest. A family of sequences is isolated. Typically, such a familycontains a common core sequence, and varying lengths of flankingsequences in different members. The shortest core sequence showingspecific binding to the antibody defines the epitope bound by theantibody. Antibodies can also be tested for epitope specificity in acompetition assay with an antibody whose epitope specificity has alreadybeen determined. For example, antibodies that compete with the 3D6antibody for binding to Aβ bind to the same or similar epitope as 3D6,i.e., within residues Aβ 1-5. Likewise antibodies that compete with the10D5 antibody bind to the same or similar epitope, i.e., within residuesAβ 3-7. Screening antibodies for epitope specificity is a usefulpredictor of therapeutic efficacy. For example, an antibody determinedto bind to an epitope within residues 1-7 of Aβ is likely to beeffective in preventing and treating Alzheimer's disease according tothe methodologies of the present invention.

[0079] Monoclonal or polyclonal antibodies that specifically bind to apreferred segment of Aβ without binding to other regions of Aβ have anumber of advantages relative to monoclonal antibodies binding to otherregions or polyclonal sera to intact Aβ. First, for equal mass dosages,dosages of antibodies that specifically bind to preferred segmentscontain a higher molar dosage of antibodies effective in clearingamyloid plaques. Second, antibodies specifically binding to preferredsegments can induce a clearing response against amyloid deposits withoutinducing a clearing response against intact APP polypeptide, therebyreducing the potential side effects.

[0080] 1. Production of Nonhuman Antibodies

[0081] The present invention features non-human antibodies, for example,antibodies having specificity for the preferred Aβ epitopes of theinvention. Such antibodies can be used in formulating varioustherapeutic compositions of the invention or, preferably, providecomplementarity determining regions for the production of humanized orchimeric antibodies (described in detail below). The production ofnon-human monoclonal antibodies, e.g., murine, guinea pig, primate,rabbit or rat, can be accomplished by, for example, immunizing theanimal with Aβ. A longer polypeptide comprising Aβ or an immunogenicfragment of Aβ or anti-idiotypic antibodies to an antibody to Aβ canalso be used. See Harlow & Lane, supra, incorporated by reference forall purposes). Such an immunogen can be obtained from a natural source,by peptide synthesis or by recombinant expression. Optionally, theimmunogen can be administered fused or otherwise complexed with acarrier protein, as described below. Optionally, the immunogen can beadministered with an adjuvant. The term “adjuvant” refers to a compoundthat when administered in conjunction with an antigen augments theimmune response to the antigen, but when administered alone does notgenerate an immune response to the antigen. Adjuvants can augment animmune response by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals.

[0082] Rabbits or guinea pigs are typically used for making polyclonalantibodies. Exemplary preparation of polyclonal antibodies, e.g., forpassive protection, can be performed as follows. 125 non-transgenic miceare immunized with 100 μg Aβ1-42, plus CFA/IFA adjuvant, and euthanizedat 4-5 months. Blood is collected from immunized mice. IgG is separatedfrom other blood components. Antibody specific for the immunogen may bepartially purified by affinity chromatography. An average of about 0.5-1mg of immunogen-specific antibody is obtained per mouse, giving a totalof 60-120 mg.

[0083] Mice are typically used for making monoclonal antibodies.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to Aβ.Optionally, antibodies are screened for binding to a specific region ordesired fragment of Aβ without binding to other nonoverlapping fragmentsof Aβ. The latter screening can be accomplished by determining bindingof an antibody to a collection of deletion mutants of an Aβ peptide anddetermining which deletion mutants bind to the antibody. Binding can beassessed, for example, by Western blot or ELISA. The smallest fragmentto show specific binding to the antibody defines the epitope of theantibody. Alternatively, epitope specificity can be determined by acompetition assay is which a test and reference antibody compete forbinding to Aβ. If the test and reference antibodies compete, then theybind to the same epitope or epitopes sufficiently proximal such thatbinding of one antibody interferes with binding of the other. Thepreferred isotype for such antibodies is mouse isotype IgG2a orequivalent isotype in other species. Mouse isotype IgG2a is theequivalent of human isotype IgG1.

[0084] 2. Chimeric and Humanized Antibodies

[0085] The present invention also features chimeric and/or humanizedantibodies (i.e., chimeric and/or humanized immunoglobulins) specificfor beta amyloid peptide. Chimeric and/or humanized antibodies have thesame or similar binding specificity and affinity as a mouse or othernonhuman antibody that provides the starting material for constructionof a chimeric or humanized antibody.

[0086] A. Production of Chimeric Antibodies

[0087] The term “chimeric antibody” refers to an antibody whose lightand heavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG1 and IgG4. Human isotype IgG1 is preferred. A typicalchimeric antibody is thus a hybrid protein consisting of the V orantigen-binding domain from a mouse antibody and the C or effectordomain from a human antibody.

[0088] B. Production of Humanized Antibodies

[0089] The term “humanized antibody” refers to an antibody comprising atleast one chain comprising variable region framework residuessubstantially from a human antibody chain (referred to as the acceptorimmunoglobulin or antibody) and at least one complementarity determiningregion substantially from a mouse-antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762. Selick etal., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin.

[0090] The substitution of mouse CDRs into a human variable domainframework is most likely to result in retention of their correct spatialorientation if the human variable domain framework adopts the same orsimilar conformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

[0091] Having identified the complementarity determining regions of themurine donor immunoglobulin and appropriate human acceptorimmunoglobulins, the next step is to determine which, if any, residuesfrom these components should be substituted to optimize the propertiesof the resulting humanized antibody. In general, substitution of humanamino acid residues with murine should be minimized, becauseintroduction of murine residues increases the risk of the antibodyeliciting a human-anti-mouse-antibody (HAMA) response in humans.Art-recognized methods of determining immune response can be performedto monitor a HAMA response in a particular patient or during clinicaltrials. Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

[0092] Certain amino acids from the human variable region frameworkresidues are selected for substitution based on their possible influenceon CDR conformation and/or binding to antigen. The unnaturaljuxtaposition of murine CDR regions with human variable framework regioncan result in unnatural conformational restraints, which, unlesscorrected by substitution of certain amino acid residues, lead to lossof binding affinity.

[0093] The selection of amino acid residues for substitution isdetermined, in part, by computer modeling. Computer hardware andsoftware are described herein for producing three-dimensional images ofimmunoglobulin molecules. In general, molecular models are producedstarting from solved structures for immunoglobulin chains or domainsthereof. The chains to be modeled are compared for amino acid sequencesimilarity with chains or domains of solved three-dimensionalstructures, and the chains or domains showing the greatest sequencesimilarity is/are selected as starting points for construction of themolecular model. Chains or domains sharing at least 50% sequenceidentity are selected for modeling, and preferably those sharing atleast 60%. 70%, 80%, 90% sequence identity or more are selected formodeling. The solved starting structures are modified to allow fordifferences between the actual amino acids in the immunoglobulin chainsor domains being modeled, and those in the starting structure. Themodified structures are then assembled into a composite immunoglobulin.Finally, the model is refined by energy minimization and by verifyingthat all atoms are within appropriate distances from one another andthat bond lengths and angles are within chemically acceptable limits.

[0094] The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

[0095] (1) noncovalently binds antigen directly,

[0096] (2) is adjacent to a CDR region,

[0097] (3) otherwise interacts with a CDR region (e.g., is within about3-6 Å of a CDR region as determined by computer modeling), or

[0098] (4) participates in the VL-VH interface.

[0099] Residues which “noncovalently bind antigen directly” includeamino acids in positions in framework regions which are have a goodprobability of directly interacting with amino acids on the antigenaccording to established chemical forces, for example, by hydrogenbonding, Van der Waals forces, hydrophobic interactions, and the like.

[0100] CDR and framework regions are as defined by Kabat et al. orChothia et al., supra. When framework residues, as defined by Kabat etal., supra, constitute structural loop residues as defined by Chothia etal., supra, the amino acids present in the mouse antibody may beselected for substitution into the humanized antibody. Residues whichare “adjacent to a CDR region” include amino acid residues in positionsimmediately adjacent to one or more of the CDRs in the primary sequenceof the humanized immunoglobulin chain, for example, in positionsimmediately adjacent to a CDR as defined by Kabat, or a CDR as definedby Chothia (See e.g., Chothia and Lesk JMB 196:901 (1987)). These aminoacids are particularly likely to interact with the amino acids in theCDRs and, if chosen from the acceptor, to distort the donor CDRs andreduce affinity. Moreover, the adjacent amino acids may interactdirectly with the antigen (Amit et al., Science, 233:747 (1986), whichis incorporated herein by reference) and selecting these amino acidsfrom the donor may be desirable to keep all the antigen contacts thatprovide affinity in the original antibody.

[0101] Residues that “otherwise interact with a CDR region” includethose that are determined by secondary structural analysis to be in aspatial orientation sufficient to effect a CDR region. In oneembodiment, residues that “otherwise interact with a CDR region” areidentified by analyzing a three-dimensional model of the donorimmunoglobulin (e.g., a computer-generated model). A three-dimensionalmodel, typically of the original donor antibody, shows that certainamino acids outside of the CDRs are close to the CDRs and have a goodprobability of interacting with amino acids in the CDRs by hydrogenbonding, Van der Waals forces, hydrophobic interactions, etc. At thoseamino acid positions, the donor immunoglobulin amino acid rather thanthe acceptor immunoglobulin amino acid may be selected. Amino acidsaccording to this criterion will generally have a side chain atom withinabout 3 angstrom units (Å) of some atom in the CDRs and must contain anatom that could interact with the CDR atoms according to establishedchemical forces, such as those listed above.

[0102] In the case of atoms that may form a hydrogen bond, the 3 Å ismeasured between their nuclei, but for atoms that do not form a bond,the 3 Å is measured between their Van der Waals surfaces. Hence, in thelatter case, the nuclei must be within about 6 Å (3 Å plus the sum ofthe Van der Waals radii) for the atoms to be considered capable ofinteracting. In many cases the nuclei will be from 4 or 5 to 6 Å apart.In determining whether an amino acid can interact with the CDRs, it ispreferred not to consider the last 8 amino acids of heavy chain CDR 2 aspart of the CDRs, because from the viewpoint of structure, these 8 aminoacids behave more as part of the framework.

[0103] Amino acids that are capable of interacting with amino acids inthe CDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

[0104] The amino acids at several positions in the framework are knownto be capable of interacting with the CDRs in many antibodies (Chothiaand Lesk, supra, Chothia et al. supra and Tramontano et al., J. Mol.Biol. 215:175 (1990), all of which are incorporated herein byreference). Notably, the amino acids at positions 2, 48, 64 and 71 ofthe light chain and 26-30, 71 and 94 of the heavy chain (numberingaccording to Kabat) are known to be capable of interacting with the CDRsin many antibodies. The amino acids at positions 35 in the light chainand 93 and 103 in the heavy chain are also likely to interact with theCDRs. At all these numbered positions, choice of the donor amino acidrather than the acceptor amino acid (when they differ) to be in thehumanized immunoglobulin is preferred. On the other hand, certainresidues capable of interacting with the CDR region, such as the first 5amino acids of the light chain, may sometimes be chosen from theacceptor immunoglobulin without loss of affinity in the humanizedimmunoglobulin.

[0105] Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

[0106] In general, one or more of the amino acids fulfilling the abovecriteria is substituted. In some embodiments, all or most of the aminoacids fulfilling the above criteria are substituted. Occasionally, thereis some ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

[0107] Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gin; ser, thr; lys, arg; and phe, tyr.

[0108] Additional candidates for substitution are acceptor humanframework amino acids that are unusual or “rare” for a humanimmunoglobulin at that position. These amino acids can be substitutedwith amino acids from the equivalent position of the mouse donorantibody or from the equivalent positions of more typical humanimmunoglobulins. For example, substitution may be desirable when theamino acid in a human framework region of the acceptor immunoglobulin israre for that position and the corresponding amino acid in the donorimmunoglobulin is common for that position in human immunoglobulinsequences; or when the amino acid in the acceptor immunoglobulin is rarefor that position and the corresponding amino acid in the donorimmunoglobulin is also rare, relative to other human sequences. Thesecriterion help ensure that an a typical amino acid in the humanframework does not disrupt the antibody structure. Moreover, byreplacing an unusual human acceptor amino acid with an amino acid fromthe donor antibody that happens to be typical for human antibodies, thehumanized antibody may be made less immunogenic.

[0109] The term “rare”, as used herein, indicates an amino acidoccurring at that position in less than about 20% but usually less thanabout 10% of sequences in a representative sample of sequences, and theterm “common”, as used herein, indicates an amino acid occurring in morethan about 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

[0110] Additional candidates for substitution are acceptor humanframework amino acids that would be identified as part of a CDR regionunder the alternative definition proposed by Chothia et al., supra.Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under theAbM and/or contact definitions. Notably, CDR1 in the variable heavychain is defined as including residues 26-32.

[0111] Additional candidates for substitution are acceptor frameworkresidues that correspond to a rare or unusual donor framework residue.Rare or unusual donor framework residues are those that are rare orunusual (as defined herein) for murine antibodies at that position. Formurine antibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding can be substituted.

[0112] Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

[0113] Other than the specific amino acid substitutions discussed above,the framework regions of humanized immunoglobulins are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human antibodies from which they were derived. Of course,many of the amino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

[0114] The humanized antibodies preferably exhibit a specific bindingaffinity for antigen of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually theupper limit of binding affinity of the humanized antibodies for antigenis within a factor of three, four or five of that of the donorimmunoglobulin. Often the lower limit of binding affinity is also withina factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized antibody having no substitutions (e.g., an antibody havingdonor CDRs and acceptor FRs, but no FR substitutions). In suchinstances, the binding of the optimized antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIACORE (i.e., surface plasmon resonance using unlabelledreagents) or competitive binding assays.

[0115] C. Production of Humanized 3D6 Antibodies

[0116] A preferred embodiment of the present invention features ahumanized antibody to the N-terminus of Aβ, in particular, for use inthe therapeutic and/or diagnostic methodologies described herein. Aparticularly preferred starting material for production of humanizedantibodies is 3D6. 3D6 is specific for the N-terminus of Aβ and has beenshown to mediate phagocytosis (e.g., induce phagocytosis) of amyloidplaque (see Examples I-V). The cloning and sequencing of cDNA encodingthe 3D6 antibody heavy and light chain variable regions is described inExample VI.

[0117] Suitable human acceptor antibody sequences are identified bycomputer comparisons of the amino acid sequences of the mouse variableregions with the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each. In particular, variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine VL and VH framework regions were identified by query ofthe Kabat Database using NCBI BLAST (publicly accessible through theNational Institutes of Health NCBI internet server) with the respectivemurine framework sequences. In one embodiment, acceptor sequencessharing greater that 50% sequence identity with murine donor sequencesare selected. Preferably, acceptor antibody sequences sharing 60%, 70%,80%, 90% or more are selected.

[0118] A computer comparison of 3D6 revealed that the 3D6 light chainshows the greatest sequence identity to human light chains of subtypekappa II, and that the 3D6 heavy chain shows greatest sequence identityto human heavy chains of subtype III, as defined by Kabat et al., supra.Thus, light and heavy human framework regions are preferably derivedfrom human antibodies of these subtypes, or from consensus sequences ofsuch subtypes. The preferred light chain human variable regions showinggreatest sequence identity to the corresponding region from 3D6 are fromantibodies having Kabat ID Numbers 019230, 005131, 005058, 005057,005059, U21040 and U41645, with 019230 being more preferred. Thepreferred heavy chain human variable regions showing greatest sequenceidentity to the corresponding region from 3D6 are from antibodies havingKabat ID Numbers 045919, 000459, 000553, 000386 and M23691, with 045919being more preferred.

[0119] Residues are next selected for substitution, as follows. When anamino acid differs between a 3D6 variable framework region and anequivalent human variable framework region, the human framework aminoacid should usually be substituted by the equivalent mouse amino acid ifit is reasonably expected that the amino acid:

[0120] (1) noncovalently binds antigen directly,

[0121] (2) is adjacent to a CDR region, is part of a CDR region underthe alternative definition proposed by Chothia et al., supra, orotherwise interacts with a CDR region (e.g., is within about 3 A of aCDR region) (e.g., amino acids at positions L2, H49 and H94 of 3D6), or

[0122] (3) participates in the VL-VH interface (e.g., amino acids atpositions L36, L46 and H93 of 3D6).

[0123] Computer modeling of the 3D6 antibody heavy and light chainvariable regions, and humanization of the 3D6 antibody is described inExample VII. Briefly, a three-dimensional model was generated based onthe closest solved murine antibody structures for the heavy and lightchains. For this purpose, an antibody designated 1 CR9 (Protein DataBank (PDB) ID: 1CR9, Kanyo et al., J. Mol. Biol. 293:855 (1999)) waschosen as a template for modeling the 3D6 light chain, and an antibodydesignated 1OPG (PDB ID: 1OPG, Kodandapani et al., J. Biol. Chem.270:2268 (1995)) was chosen as the template for modeling the heavychain. The model was further refined by a series of energy minimizationsteps to relieve unfavorable atomic contacts and optimize electrostaticand van der Walls interactions. The solved structure of 1qkz (PDB ID:1QKZ, Derrick et al., J. Mol. Biol. 293:81 (1999)) was chosen as atemplate for modeling CDR3 of the heavy chain as 3D6 and 1OPG did notexhibit significant sequence homology in this region when aligned forcomparison purposes.

[0124] Three-dimensional structural information for the antibodiesdescribed herein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, 28:235.Computer modeling allows for the identification of CDR-interactingresidues. The computer model of the structure of 3D6 can in turn serveas a starting point for predicting the three-dimensional structure of anantibody containing the 3D6 complementarity determining regionssubstituted in human framework structures. Additional models can beconstructed representing the structure as further amino acidsubstitutions are introduced.

[0125] In general, substitution of one, most or all of the amino acidsfulfilling the above criteria is desirable. Accordingly, the humanizedantibodies of the present invention will usually contain a substitutionof a human light chain framework residue with a corresponding 3D6residue in at least 1, 2 or 3, and more usually 4, of the followingpositions: L1, L2, L36 and L46. The humanized antibodies also usuallycontain a substitution of a human heavy chain framework residue with acorresponding 3D6 residue in at least 1, 2, and sometimes 3, of thefollowing positions: H49, H93 and H94. Humanized antibodies can alsocontain a substitution of a heavy chain framework residue with acorresponding germline residue in at least 1, 2, and sometimes 3, of thefollowing positions: H74, H77 and H89.

[0126] Occasionally, however, there is some ambiguity about whether aparticular amino acid meets the above criteria, and alternative variantimmunoglobulins are produced, one of which has that particularsubstitution, the other of which does not. In instances wheresubstitution with a murine residue would introduce a residue that israre in human immunoglobulins at a particular position, it may bedesirable to test the antibody for activity with or without theparticular substitution. If activity (e.g., binding affinity and/orbinding specificity) is about the same with or without the substitution,the antibody without substitution may be preferred, as it would beexpected to elicit less of a HAHA response, as described herein.

[0127] Other candidates for substitution are acceptor human frameworkamino acids that are unusual for a human immunoglobulin at thatposition. These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouse 3D6can be introduced into the human framework regions when such amino acidsare typical of human immunoglobulin at the equivalent positions.

[0128] In additional embodiments, when the human light chain frameworkacceptor immunoglobulin is Kabat ID Number 019230, the light chaincontains substitutions in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more usually 13, of the following positions: L7, L10, L12, L15, L17,L39, L45, L63, L78, L83, L85, L100 or L104. In additional embodimentswhen the human heavy chain framework acceptor immunoglobulin is Kabat IDNumber 045919, the heavy chain contains substitutions in at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more usually 15, of thefollowing positions: H3, H5, H13, H16, H19, H40, H41, H42, H44, H72,H77, H82A, H83, H84, or H108. These positions are substituted with theamino acid from the equivalent position of a human immunoglobulin havinga more typical amino acid residue. Examples of appropriate amino acidsto substitute are shown in FIGS. 1 and 2.

[0129] Other candidates for substitution are non-germline residuesoccurring in a framework region. A computer comparison of 3D6 with knowngermline sequences revealed that heavy chains showing the greatestdegree of sequence identity include germline variable region sequencesVH3-48, VH3-23, VH3-7, VH3-21 and VH3-11, with VH3-23 being morepreferred. Alignment of Kabat ID 045919 with VH3-23 reveals thatresidues H74, H77 and/or H89 may be selected for substitution withcorresponding germline residues (e.g., residues H74, H77 and/or H89 whencomparing Kabat ID 045919 and VH3-23). Likewise, germline sequenceshaving the greatest degree of identity to the 3D6 light chain includeA1, A 17, A18, A2 and A 19, with A19 being most preferred. Residues notmatching between a selected light chain acceptor framework and one ofthese germline sequences could be selected for substitution with thecorresponding germline residue.

[0130] Table 1 summarizes the sequence analysis of the 3D6 VH and VLregions. Additional mouse and human structures that can be used forcomputer modeling of the 3D6 antibody and additional human antibodiesare set forth as well as germline sequences that can be used inselecting amino acid substitutions. Rare mouse residues are also setforth in Table 1. Rare mouse residues are identified by comparing thedonor VL and/or VH sequences with the sequences of other members of thesubgroup to which the donor VL and/or VH sequences belong (according toKabat) and identifying the residue positions which differ from theconsensus. These donor specific differences may point to somaticmutations which enhance activity. Unusual or rare residues close to thebinding site may possibly contact the antigen, making it desirable toretain the mouse residue. However, if the unusual mouse residue is notimportant for binding, use of the corresponding acceptor residue ispreferred as the mouse residue may create immunogenic neoepitopes in thehumanized antibody. In the situation where an unusual residue in thedonor sequence is actually a common residues in the correspondingacceptor sequence, the preferred residue is clearly the acceptorresidue. TABLE 1 Summary of 3D6 V-region sequence Chain Heavy LightMouse subgroup IIID (002688) II (005840-005844, 005851-005853, (Kabatseq ID#) 005857, 005863) Mouse homologs 002727/163.1′CL 005840/1210.7(Kabat/Genbank) 002711/H35-C6′CL 005843/42.4b.12.2′CL002733/8-1-12-5-3-1(A2-1)′CL 005842/BXW-14′CL 002715/ASWA2′CL005841/42.7B3.2′CL 020669/#14′CL 005851/36-60CRI- Rare amino acids (%N40 (0.233%) Y1(.035%) frequency of D42 (0.699%) I15 (3.3%) occurrencein class) D27 (0.867%)-CDR1 I78 (0.677%) L85 (0.625%) W89 (0.815%)-CDR3K106A (0.295%) Human Subgroup III (000488-000491, 000503, 000624) II(005046) Chothia canonical H1: class 1 [2fbj] L1: class 4 [1rmf] CDRgroupings [pdb H2: class 3 [1igc] L2: class1 [1lmk] example] L3: class 1[1tet] Closest solved mouse PDB ID: 1OPG Kodandapani et al., PDB ID:1CR9; Kanyo et al., supra; structures supra; (72% 2 Å) (94%, 2 Å) PDBID: 1NLD; Davies et al., Acta Crystallogr. D. Biol. Crystallog. 53: 186(1997); (98%, 2.8 Å) Closest solved human 1VH (68%, nmr) 1LVE (57%, LEN)structures 443560 (65%, IgG, λ myeloma, 1.8 Å) 1B6DA (54%, B-J dimer,2.8 Å); KOL/2FB4H (60%, myeloma, 3 Å) 1VGEL (54%, autoAb) Germline query(Hu) VH3-48 (4512283/BAA75032.1) A1(x63402) results (top 4) VH3-23(4512287/BAA75046.1) A17 (x63403) VH3-7 (4512300/BAA75056.1) A18(X63396) VH3-21 (4512287/BAA75047.1) A2 (m31952) VH3-11(4152300/BAA75053.1) A19 (x63397)

[0131] Kabat ID sequences referenced herein are publicly available, forexample, from the Northwestern University Biomedical EngineeringDepartment's Kabat Database of Sequences of Proteins of ImmunologicalInterest. Three-dimensional structural information for antibodiesdescribed herein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, p235-242.Germline gene sequences referenced herein are publicly available, forexample, from the National Center for Biotechnology Information (NCBI)database of sequences in collections of Igh, Ig kappa and Ig lambdagermline V genes (as a division of the National Library of Medicine(NLM) at the National Institutes of Health (NIH)). Homology searching ofthe NCBI “Ig Germline Genes” database is provided by IgG BLAST™.

[0132] In a preferred embodiment, a humanized antibody of the presentinvention contains (i) a light chain comprising a variable domaincomprising murine 3D6 VL CDRs and a human acceptor framework, theframework having at least one, preferably two, three or four residuesselected from the group consisting of L1, L2, L36, and L46 substitutedwith the corresponding 3D6 residue and (ii) a heavy chain comprising 3D6VH CDRs and a human acceptor framework, the framework having at leastone, preferably two or three residues selected from the group consistingof H49, H93 and H94 substituted with the corresponding 3D6 residue, and,optionally, at least one, preferably two or three residues selected fromthe group consisting of H74, H77 and H89 is substituted with acorresponding human germline residue.

[0133] In a more preferred embodiment, a humanized antibody of thepresent invention contains (i) a light chain comprising a variabledomain comprising murine 3D6 VL CDRs and a human acceptor framework, theframework having residue 1 substituted with a tyr (Y), residue 2substituted with a val (V), residue 36 substituted with a leu (L) and/orresidue 46 substituted with an arg (R), and (ii) a heavy chaincomprising 3D6 VH CDRs and a human acceptor framework, the frameworkhaving residue 49 substituted with an ala (A), residue 93 substitutedwith a val (V) and/or residue 94 substituted with an arg (R), and,optionally, having residue 74 substituted with a ser (S), residue 77substituted with a thr (T) and/or residue 89 substituted with a val (V).

[0134] In a particularly preferred embodiment, a humanized antibody ofthe present invention has structural features, as described herein, andfurther has at least one (preferably two, three, four or all) of thefollowing activities: (1) binds aggregated Aβ1-42 (e.g., as determinedby ELISA); (2) binds Aβ in plaques (e.g., staining of AD and/or PDAPPplaques); (3) binds Aβ with two- to three-fold higher binding affinityas compared to chimeric 3D6 (e.g., 3D6 having murine variable regionsequences and human constant region sequences); (4) mediatesphagocytosis of Aβ (e.g., in an ex vivo phagocytosis assay, as describedherein); and (5) crosses the blood-brain barrier (e.g., demonstratesshort-term brain localization, for example, in a PDAPP animal model, asdescribed herein).

[0135] In another embodiment, a humanized antibody of the presentinvention has structural features, as described herein, binds Aβ in amanner or with an affinity sufficient to elicit at least one of thefollowing in vivo effects: (1) reduce Aβ plaque burden; (2) preventplaque formation; (3) reduce levels of soluble Aβ; (4) reduce theneuritic pathology associated with an amyloidogenic disorder; (5)lessens or ameliorate at least one physiological symptom associated withan amyloidogenic disorder; and/or (6) improves cognitive function.

[0136] In another embodiment, a humanized antibody of the presentinvention has structural features, as described herein, and specificallybinds to an epitope comprising residues 1-5 or 3-7 of Aβ.

[0137] The activities described above can be determined utilizing anyone of a variety of assays described herein or in the art (e.g., bindingassays, phagocytosis assays, etc.). Activities can be assayed either invivo (e.g., using labeled assay components and/or imaging techniques) orin vitro (e.g., using samples or specimens derived from a subject).Activities can be assayed either directly or indirectly. In certainpreferred embodiments, neurological endpoints (e.g., amyloid burden,neuritic burden, etc) are assayed. Such endpoints can be assayed inliving subjects (e.g., in animal models of Alzheimer's disease or inhuman subjects, for example, undergoing immunotherapy) usingnon-invasive detection methodologies. Alternatively, such endpoints canbe assayed in subjects post mortem. Assaying such endpoints in animalmodels and/or in human subjects post mortem is useful in assessing theeffectiveness of various agents (e.g., humanized antibodies) to beutilized in similar immunotherapeutic applications. In other preferredembodiments, behavioral or neurological parameters can be assessed asindicators of the above neuropathological activities or endpoints.

[0138] 3. Human Antibodies

[0139] Human antibodies against Aβ are provided by a variety oftechniques described below. Some human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same epitopespecificity as a particular mouse antibody, such as one of the mousemonoclonals described herein. Human antibodies can also be screened fora particular epitope specificity by using only a fragment of Aβ as theimmunogen, and/or by screening antibodies against a collection ofdeletion mutants of Aβ. Human antibodies preferably have human IgG1isotype specificity.

[0140] a. Trioma Methodology

[0141] The basic approach and an exemplary cell fusion partner, SPAZ-4,for use in this approach have been described by Oestberg et al.,Hybridoma 2:361 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Englemanet al., U.S. Pat. No. 4,634,666 (each of which is incorporated byreference in its entirety for all purposes). The antibody-producing celllines obtained by this method are called triomas, because they aredescended from three cells; two human and one mouse. Initially, a mousemyeloma line is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

[0142] The immunized B-lymphocytes are obtained from the blood, spleen,lymph nodes or bone marrow of a human donor. If antibodies against aspecific antigen or epitope are desired, it is preferable to use thatantigen or epitope thereof for immunization. Immunization can be eitherin vivo or in vitro. For in vivo immunization, B cells are typicallyisolated from a human immunized with Aβ, a fragment thereof, largerpolypeptide containing Aβ or fragment, or an anti-idiotypic antibody toan antibody to Aβ. In some methods, B cells are isolated from the samepatient who is ultimately to be administered antibody therapy. For invitro immunization, B-lymphocytes are typically exposed to antigen for aperiod of 7-14 days in a media such as RPMI-1640 (see Engleman, supra)supplemented with 10% human plasma.

[0143] The immunized B-lymphocytes are fused to a xenogeneic hybrid cellsuch as SPAZ-4 by well-known methods. For example, the cells are treatedwith 40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C.,for about 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto Aβ or a fragment thereof. Triomas producing human antibodies havingthe desired specificity are subcloned by the limiting dilution techniqueand grown in vitro in culture medium. The trioma cell lines obtained arethen tested for the ability to bind Aβ or a fragment thereof.

[0144] Although triomas are genetically stable they do not produceantibodies at very high levels. Expression levels can be increased bycloning antibody genes from the trioma into one or more expressionvectors, and transforming the vector into standard mammalian, bacterialor yeast cell lines.

[0145] b. Transgenic Non-Human Mammals

[0146] Human antibodies against Aβ can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/12227 (1993);U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat.No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148:1547 (1994),Nature Biotechnology 14:826 (1996), Kucherlapati, WO 91/10741 (1991)(each of which is incorporated by reference in its entirety for allpurposes). Transgenic mice are particularly suitable. Anti-Aβ antibodiesare obtained by immunizing a transgenic nonhuman mammal, such asdescribed by Lonberg or Kucherlapati, supra, with Aβ or a fragmentthereof. Monoclonal antibodies are prepared by, e.g., fusing B-cellsfrom such mammals to suitable myeloma cell lines using conventionalKohler-Milstein technology. Human polyclonal antibodies can also beprovided in the form of serum from humans immunized with an immunogenicagent. Optionally, such polyclonal antibodies can be concentrated byaffinity purification using Aβ or other amyloid peptide as an affinityreagent.

[0147] c. Phage Display Methods

[0148] A further approach for obtaining human anti-Aβ antibodies is toscreen a DNA library from human B cells according to the generalprotocol outlined by Huse et al., Science 246:1275-1281 (1989). Asdescribed for trioma methodology, such B cells can be obtained from ahuman immunized with Aβ, fragments, longer polypeptides containing Aβ orfragments or anti-idiotypic antibodies. Optionally, such B cells areobtained from a patient who is ultimately to receive antibody treatment.Antibodies binding to Aβ or a fragment thereof are selected. Sequencesencoding such antibodies (or a binding fragments) are then cloned andamplified. The protocol described by Huse is rendered more efficient incombination with phage-display technology. See, e.g., Dower et al., WO91/17271, McCafferty et al., WO 92/01047, Herzig et al., U.S. Pat. No.5,877,218, Winter et al., U.S. Pat. No. 5,871,907, Winter et al., U.S.Pat. No. 5,858,657, Holliger et al., U.S. Pat. No. 5,837,242, Johnson etal., U.S. Pat. No. 5,733,743 and Hoogenboom et al., U.S. Pat. No.5,565,332 (each of which is incorporated by reference in its entiretyfor all purposes). In these methods, libraries of phage are produced inwhich members display different antibodies on their outer surfaces.Antibodies are usually displayed as Fv or Fab fragments. Phagedisplaying antibodies with a desired specificity are selected byaffinity enrichment to an Aβ peptide or fragment thereof.

[0149] In a variation of the phage-display method, human antibodieshaving the binding specificity of a selected murine antibody can beproduced. See Winter, WO 92/20791. In this method, either the heavy orlight chain variable region of the selected murine antibody is used as astarting material. If, for example, a light chain variable region isselected as the starting material, a phage library is constructed inwhich members display the same light chain variable region (i.e., themurine starting material) and a different heavy chain variable region.The heavy chain variable regions are obtained from a library ofrearranged human heavy chain variable regions. A phage showing strongspecific binding for Aβ (e.g., at least 10⁸ and preferably at least 10⁹M⁻¹) is selected. The human heavy chain variable region from this phagethen serves as a starting material for constructing a further phagelibrary. In this library, each phage displays the same heavy chainvariable region (i.e., the region identified from the first displaylibrary) and a different light chain variable region. The light chainvariable regions are obtained from a library of rearranged humanvariable light chain regions. Again, phage showing strong specificbinding for Aβ are selected. These phage display the variable regions ofcompletely human anti-Aβ antibodies. These antibodies usually have thesame or similar epitope specificity as the murine starting material.

[0150] 4. Production of Variable Regions

[0151] Having conceptually selected the CDR and framework components ofhumanized immunoglobulins, a variety of methods are available forproducing such immunoglobulins. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each immunoglobulin aminoacid sequence. The desired nucleic acid sequences can be produced by denovo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared variant of the desired polynucleotide. Oligonucleotide-mediatedmutagenesis is a preferred method for preparing substitution, deletionand insertion variants of target polypeptide DNA. See Adelman et al.,DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered byhybridizing an oligonucleotide encoding the desired mutation to asingle-stranded DNA template. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat incorporates the oligonucleotide primer, and encodes the selectedalteration in the target polypeptide DNA.

[0152] 5. Selection of Constant Regions

[0153] The variable segments of antibodies produced as described supra(e.g., the heavy and light chain variable regions of chimeric,humanized, or human antibodies) are typically linked to at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. Human constant region DNA sequences can beisolated in accordance with well known procedures from a variety ofhuman cells, but preferably immortalized B cells (see Kabat et al.,supra, and Liu et al., WO87/02671) (each of which is incorporated byreference in its entirety for all purposes). Ordinarily, the antibodywill contain both light chain and heavy chain constant regions. Theheavy chain constant region usually includes CH1, hinge, CH2, CH3, andCH4 regions. The antibodies described herein include antibodies havingall types of constant regions, including IgM, IgG, IgD, IgA and IgE, andany isotype, including IgG1, IgG2, IgG3 and IgG4. The choice of constantregion depends, in part, whether antibody-dependent complement and/orcellular mediated toxicity is desired. For example, isotopes IgG 1 andIgG3 have complement activity and isotypes IgG2 and IgG4 do not. When itis desired that the antibody (e.g., humanized antibody) exhibitcytotoxic activity, the constant domain is usually a complement fixingconstant domain and the class is typically IgG1. When such cytotoxicactivity is not desirable, the constant domain may be of the IgG2 class.Choice of isotype can also affect passage of antibody into the brain.Human isotype IgG1 is preferred. Light chain constant regions can belambda or kappa. The humanized antibody may comprise sequences from morethan one class or isotype. Antibodies can be expressed as tetramerscontaining two light and two heavy chains, as separate heavy chains,light chains, as Fab, Fab′ F(ab′)2, and Fv, or as single chainantibodies in which heavy and light chain variable domains are linkedthrough a spacer.

[0154] 6. Expression of Recombinant Antibodies

[0155] Chimeric, humanized and human antibodies are typically producedby recombinant expression. Nucleic acids encoding light and heavy chainvariable regions, optionally linked to constant regions, are insertedinto expression vectors. The light and heavy chains can be cloned in thesame or different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences, and the collectionand purification of the crossreacting antibodies.

[0156] These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markers(e.g., ampicillin-resistance, hygromycin-resistance, tetracyclineresistance or neomycin resistance) to permit detection of those cellstransformed with the desired DNA sequences (see, e.g., Itakura et al.,U.S. Pat. No. 4,704,362).

[0157]E. coli is one prokaryotic host particularly useful for cloningthe polynucleotides (e.g., DNA sequences) of the present invention.Other microbial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

[0158] Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

[0159] In addition to microorganisms, mammalian tissue cell culture mayalso be used to express and produce the polypeptides of the presentinvention (e.g., polynucleotides encoding immunoglobulins or fragmentsthereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y.,N.Y. (1987). Eukaryotic cells are actually preferred, because a numberof suitable host cell lines capable of secreting heterologous proteins(e.g., intact immunoglobulins) have been developed in the art, andinclude CHO cell lines, various Cos cell lines, HeLa cells, preferably,myeloma cell lines, or transformed B-cells or hybridomas. Preferably,the cells are nonhuman. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)),and necessary processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences. Preferred expression control sequences arepromoters derived from immunoglobulin genes, SV40, adenovirus, bovinepapilloma virus, cytomegalovirus and the like. See Co et al., J.Immunol. 148:1149 (1992).

[0160] Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

[0161] The vectors containing the polynucleotide sequences of interest(e.g., the heavy and light chain encoding sequences and expressioncontrol sequences) can be transferred into the host cell by well-knownmethods, which vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics or viral-based transfection may be used forother cellular hosts. (See generally Sambrook et al., Molecular Cloning:A Laboratory A Manual (Cold Spring Harbor Press, 2nd ed., 1989)(incorporated by reference in its entirety for all purposes). Othermethods used to transform mammalian cells include the use of polybrene,protoplast fusion, liposomes, electroporation, and microinjection (seegenerally, Sambrook et al., supra). For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes, or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

[0162] When heavy and light chains are cloned on separate expressionvectors, the vectors are co-transfected to obtain expression andassembly of intact immunoglobulins. Once expressed, the wholeantibodies, their dimers, individual light and heavy chains, or otherimmunoglobulin forms of the present invention can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, HPLCpurification, gel electrophoresis and the like (see generally Scopes,Protein Purification (Springer-Verlag, N.Y., (1982)). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred, for pharmaceuticaluses.

[0163] 7. Antibody Fragments

[0164] Also contemplated within the scope of the instant invention areantibody fragments. In one embodiment, fragments of non-human, chimericand/or human antibodies are provided. In another embodiment, fragmentsof humanized antibodies are provided. Typically, these fragments exhibitspecific binding to antigen with an affinity of at least 10⁷, and moretypically 10⁸ or 10⁹ M⁻¹. Humanized antibody fragments include separateheavy chains, light chains Fab, Fab′F(ab′)2, Fabc, and Fv. Fragments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

[0165] 8. Testing Antibodies for Therapeutic Efficacy in Animal Models

[0166] Groups of 7-9 month old PDAPP mice each are injected with 0.5 mgin PBS of polyclonal anti-Aβ or specific anti-Aβ monoclonal antibodies.All antibody preparations are purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.

[0167] Mice are injected intraperitoneally as needed over a 4 monthperiod to maintain a circulating antibody concentration measured byELISA titer of greater than {fraction (1/1000)} defined by ELISA to Aβ42or other immunogen. Titers are monitored and mice are euthanized at theend of 6 months of injections. Histochemistry, Aβ levels and toxicologyare performed post mortem. Ten mice are used per group.

[0168] 9. Screening Antibodies for Clearing Activity

[0169] The invention also provides methods of screening an antibody foractivity in clearing an amyloid deposit or any other antigen, orassociated biological entity, for which clearing activity is desired. Toscreen for activity against an amyloid deposit, a tissue sample from abrain of a patient with Alzheimer's disease or an animal model havingcharacteristic Alzheimer's pathology is contacted with phagocytic cellsbearing an Fc receptor, such as microglial cells, and the antibody undertest in a medium in vitro. The phagocytic cells can be a primary cultureor a cell line, such as BV-2, C8-B4, or THP-1. In some methods, thecomponents are combined on a microscope slide to facilitate microscopicmonitoring. In some methods, multiple reactions are performed inparallel in the wells of a microtiter dish. In such a format, a separateminiature microscope slide can be mounted in the separate wells, or anonmicroscopic detection format, such as ELISA detection of Aβ can beused. Preferably, a series of measurements is made of the amount ofamyloid deposit in the in vitro reaction mixture, starting from abaseline value before the reaction has proceeded, and one or more testvalues during the reaction. The antigen can be detected by staining, forexample, with a fluorescently labeled antibody to Aβ or other componentof amyloid plaques. The antibody used for staining may or may not be thesame as the antibody being tested for clearing activity. A reductionrelative to baseline during the reaction of the amyloid depositsindicates that the antibody under test has clearing activity. Suchantibodies are likely to be useful in preventing or treating Alzheimer'sand other amyloidogenic diseases.

[0170] Analogous methods can be used to screen antibodies for activityin clearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used include Aβ,viral antigens or viruses, proteoglycans, antigens of other pathogenicmicroorganisms, tumor antigens, and adhesion molecules. Such antigenscan be obtained from natural sources, recombinant expression or chemicalsynthesis, among other means. The tissue sample or isolated biologicalentity is contacted with phagocytic cells bearing Fc receptors, such asmonocytes or microglial cells, and an antibody to be tested in a medium.The antibody can be directed to the biological entity under test or toan antigen associated with the entity. In the latter situation, theobject is to test whether the biological entity is vicariouslyphagocytosed with the antigen. Usually, although not necessarily, theantibody and biological entity (sometimes with an associated antigen),are contacted with each other before adding the phagocytic cells. Theconcentration of the biological entity and/or the associated antigenremaining in the medium, if present, is then monitored. A reduction inthe amount or concentration of antigen or the associated biologicalentity in the medium indicates the antibody has a clearing responseagainst the antigen and/or associated biological entity in conjunctionwith the phagocytic cells (see, e.g., Example IV).

[0171] 10. Chimeric/Humanized Antibodies Having Altered EffectorFunction

[0172] For the above-described antibodies of the invention comprising aconstant region (Fc region), it may also be desirable to alter theeffector function of the molecule. Generally, the effector function ofan antibody resides in the constant or Fc region of the molecule whichcan mediate binding to various effector molecules, e.g., complementproteins or Fc receptors. The binding of complement to the Fc region isimportant, for example, in the opsonization and lysis of cell pathogensand the activation of inflammatory responses. The binding of antibody toFc receptors, for example, on the surface of effector cells can triggera number of important and diverse biological responses including, forexample, engulfment and destruction of antibody-coated pathogens orparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (i.e., antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer of antibodies, and control of immunoglobulin production.

[0173] Accordingly, depending on a particular therapeutic or diagnosticapplication, the above-mentioned immune functions, or only selectedimmune functions, may be desirable. By altering the Fc region of theantibody, various aspects of the effector function of the molecule,including enhancing or suppressing various reactions of the immunesystem, with beneficial effects in diagnosis and therapy, are achieved.

[0174] The antibodies of the invention can be produced which react onlywith certain types of Fc receptors, for example, the antibodies of theinvention can be modified to bind to only certain Fc receptors, or ifdesired, lack Fc receptor binding entirely, by deletion or alteration ofthe Fc receptor binding site located in the Fc region of the antibody.Other desirable alterations of the Fc region of an antibody of theinvention are cataloged below. Typically the Kabat numbering system isused to indicate which amino acid residue(s) of the Fc region (e.g., ofan IgG antibody) are altered (e.g., by amino acid substitution) in orderto achieve a desired change in effector function. The numbering systemis also employed to compare antibodies across species such that adesired effector function observed in, for example, a mouse antibody,can then be systematically engineered into a human, humanized, orchimeric antibody of the invention.

[0175] For example, it has been observed that antibodies (e.g., IgGantibodies) can be grouped into those found to exhibit tight,intermediate, or weak binding to an Fc receptor (e.g., an Fc receptor onhuman monocytes (FcγRI)). By comparison of the amino-acid sequences inthese different affinity groups, a monocyte-binding site in thehinge-link region (Leu234-Ser239) has been identified. Moreover, thehuman FcγRI receptor binds human IgG1 and mouse IgG2a as a monomer, butthe binding of mouse IgG2b is 100-fold weaker. A comparison of thesequence of these proteins in the hinge-link region shows that thesequence 234 to 238, i.e., Leu-Leu-Gly-Gly-Pro in the strong bindersbecomes Leu-Glu-Gly-Gly-Pro in mouse gamma 2b, i.e., weak binders.Accordingly, a corresponding change in a human antibody hinge sequencecan be made if reduced FcγI receptor binding is desired. It isunderstood that other alterations can be made to achieve the same orsimilar results. For example, the affinity of FcγRI binding can bealtered by replacing the specified residue with a residue having aninappropriate functional group on its sidechain, or by introducing acharged functional group (e.g., Glu or Asp) or for example an aromaticnon-polar residue (e.g., Phe, Tyr, or Trp).

[0176] These changes can be equally applied to the murine, human, andrat systems given the sequence homology between the differentimmunoglobulins. It has been shown that for human IgG3, which binds tothe human FcγRI receptor, changing Leu 235 to Glu destroys theinteraction of the mutant for the receptor. The binding site for thisreceptor can thus be switched on or switched off by making theappropriate mutation.

[0177] Mutations on adjacent or close sites in the hinge link region(e.g., replacing residues 234, 236 or 237 by Ala) indicate thatalterations in residues 234, 235, 236, and 237 at least affect affinityfor the FcγRI receptor. Accordingly, the antibodies of the invention canalso have an altered Fc region with altered binding affinity for FcγRIas compared with the unmodified antibody. Such an antibody convenientlyhas a modification at amino acid residue 234, 235, 236, or 237.

[0178] Affinity for other Fc receptors can be altered by a similarapproach, for controlling the immune response in different ways.

[0179] As a further example, the lytic properties of IgG antibodiesfollowing binding of the Cl component of complement can be altered.

[0180] The first component of the complement system, Cl, comprises threeproteins known as Clq, Clr and Cls which bind tightly together. It hasbeen shown that Clq is responsible for binding of the three proteincomplex to an antibody.

[0181] Accordingly, the Clq binding activity of an antibody can bealtered by providing an antibody with an altered CH 2 domain in which atleast one of the amino acid residues 318, 320, and 322 of the heavychain has been changed to a residue having a different side chain. Thenumbering of the residues in the heavy chain is that of the EU index(see Kabat et al., supra). Other suitable alterations for altering,e.g., reducing or abolishing specific Clq-binding to an antibody includechanging any one of residues 318 (Glu), 320 (Lys) and 322 (Lys), to Ala.

[0182] Moreover, by making mutations at these residues, it has beenshown that Clq binding is retained as long as residue 318 has ahydrogen-bonding side chain and residues 320 and 322 both have apositively charged side chain.

[0183] Clq binding activity can be abolished by replacing any one of thethree specified residues with a residue having an inappropriatefunctionality on its side chain. It is not necessary to replace theionic residues only with Ala to abolish Clq binding. It is also possibleto use other alkyl-substituted non-ionic residues, such as Gly, Ile,Leu, or Val, or such aromatic non-polar residues as Phe, Tyr, Trp andPro in place of any one of the three residues in order to abolish Clqbinding. In addition, it is also be possible to use such polar non-ionicresidues as Ser, Thr, Cys, and Met in place of residues 320 and 322, butnot 318, in order to abolish Clq binding activity.

[0184] It is also noted that the side chains on ionic or non-ionic polarresidues will be able to form hydrogen bonds in a similar manner to thebonds formed by the Glu residue. Therefore, replacement of the 318 (Glu)residue by a polar residue may modify but not abolish Clq bindingactivity.

[0185] It is also known that replacing residue 297 (Asn) with Alaresults in removal of lytic activity while only slightly reducing (aboutthree fold weaker) affinity for Clq. This alteration destroys theglycosylation site and the presence of carbohydrate that is required forcomplement activation. Any other substitution at this site will alsodestroy the glycosylation site.

[0186] The invention also provides an antibody having an alteredeffector function wherein the antibody has a modified hinge region. Themodified hinge region may comprise a complete hinge region derived froman antibody of different antibody class or subclass from that of the CH1domain. For example, the constant domain (CH1) of a class IgG antibodycan be attached to a hinge region of a class IgG4 antibody.Alternatively, the new hinge region may comprise part of a natural hingeor a repeating unit in which each unit in the repeat is derived from anatural hinge region. In one example, the natural hinge region isaltered by converting one or more cysteine residues into a neutralresidue, such as alanine, or by converting suitably placed residues intocysteine residues. Such alterations are carried out using art recognizedprotein chemistry and, preferably, genetic engineering techniques, asdescribed herein.

[0187] In one embodiment of the invention, the number of cysteineresidues in the hinge region of the antibody is reduced, for example, toone cysteine residue. This modification has the advantage offacilitating the assembly of the antibody, for example, bispecificantibody molecules and antibody molecules wherein the Fc portion hasbeen replaced by an effector or reporter molecule, since it is onlynecessary to form a single disulfide bond. This modification alsoprovides a specific target for attaching the hinge region either toanother hinge region or to an effector or reporter molecule, eitherdirectly or indirectly, for example, by chemical means.

[0188] Conversely, the number of cysteine residues in the hinge regionof the antibody is increased, for example, at least one more than thenumber of normally occurring cysteine residues. Increasing the number ofcysteine residues can be used to stabilize the interactions betweenadjacent hinges. Another advantage of this modification is that itfacilitates the use of cysteine thiol groups for attaching effector orreporter molecules to the altered antibody, for example, a radiolabel.

[0189] Accordingly, the invention provides for an exchange of hingeregions between antibody classes, in particular, IgG classes, and/or anincrease or decrease in the number of cysteine residues in the hingeregion in order to achieve an altered effector function (see for exampleU.S. Pat. No. 5,677,425 which is expressly incorporated herein). Adetermination of altered antibody effector function is made using theassays described herein or other art recognized techniques.

[0190] Importantly, the resultant antibody can be subjected to one ormore assays to evaluate any change in biological activity compared tothe starting antibody.

[0191] For example, the ability of the antibody with an altered Fcregion to bind complement or Fc receptors can be assessed using theassays disclosed herein as well as any art recognized assay.

[0192] Production of the antibodies of the invention is carried out byany suitable technique including techniques described herein as well astechniques known to those skilled in the art. For example an appropriateprotein sequence, e.g. forming part of or all of a relevant constantdomain, e.g., Fc region, i.e., CH2, and/or CH3 domain(s), of anantibody, and include appropriately altered residue(s) can besynthesized and then chemically joined into the appropriate place in anantibody molecule.

[0193] Preferably, genetic engineering techniques are used for producingan altered antibody. Preferred techniques include, for example,preparing suitable primers for use in polymerase chain reaction (PCR)such that a DNA sequence which encodes at least part of an IgG heavychain, e.g., an Fc or constant region (e.g., CH2, and/or CH3) isaltered, at one or more residues. The segment can then be operablylinked to the remaining portion of the antibody, e.g., the variableregion of the antibody and required regulatory elements for expressionin a cell.

[0194] The present invention also includes vectors used to transform thecell line, vectors used in producing the transforming vectors, celllines transformed with the transforming vectors, cell lines transformedwith preparative vectors, and methods for their production.

[0195] Preferably, the cell line which is transformed to produce theantibody with an altered Fc region (i.e., of altered effector function)is an immortalized mammalian cell line (e.g., CHO cell).

[0196] Although the cell line used to produce the antibody with analtered Fc region is preferably a mammalian cell line, any othersuitable cell line, such as a bacterial cell line or a yeast cell line,may alternatively be used.

[0197] B. Nucleic Acid Encoding Immunologic and Therapeutic Agents

[0198] Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding antibodies and their componentchains used for passive immunization. Such nucleic acids can be DNA orRNA. A nucleic acid segment encoding an immunogen is typically linked toregulatory elements, such as a promoter and enhancer, that allowexpression of the DNA segment in the intended target cells of a patient.For expression in blood cells, as is desirable for induction of animmune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors.

[0199] A number of viral vector systems are available includingretroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet.Develop. 3:102-109 (1993)); adenoviral vectors (see, e.g., Bett et al.,J. Virol. 67:5911 (1993)); adeno-associated virus vectors (see, e.g.,Zhou et al., J. Exp. Med. 179:1867 (1994)), viral vectors from the poxfamily including vaccinia virus and the avian pox viruses, viral vectorsfrom the alpha virus genus such as those derived from Sindbis andSemliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70:508(1996)), Venezuelan equine encephalitis virus (see Johnston et al., U.S.Pat. No. 5,643,576) and rhabdoviruses, such as vesicular stomatitisvirus (see Rose, WO 96/34625) and papillomaviruses (Ohe et al., HumanGene Therapy 6:325 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res. 24, 2630-2622 (1996)).

[0200] DNA encoding an immunogen, or a vector containing the same, canbe packaged into liposomes. Suitable lipids and related analogs aredescribed by Eppstein et al., U.S. Pat. No. 5,208,036, Felgner et al.,U.S. Pat. No. 5,264,618, Rose, U.S. Pat. No. 5,279,833, and Epand etal., U.S. Pat. No. 5,283,185. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

[0201] Gene therapy vectors or naked polypeptides (e.g., DNA) can bedelivered in vivo by administration to an individual patient, typicallyby systemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranialinfusion) or topical application (see e.g., Anderson et al., U.S. Pat.No. 5,399,346). The term “naked polynucleotide” refers to apolynucleotide not complexed with colloidal materials. Nakedpolynucleotides are sometimes cloned in a plasmid vector. Such vectorscan further include facilitating agents such as bupivacine (Attardo etal., U.S. Pat. No. 5,593,970). DNA can also be administered using a genegun. See Xiao & Brandsma, supra. The DNA encoding an immunogen isprecipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see Howell et al., WO 95/05853).

[0202] In a further variation, vectors encoding immunogens can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which haveincorporated the vector.

[0203] II. Prophylactic and Therapeutic Methods

[0204] The present invention is directed inter alia to treatment ofAlzheimer's and other amyloidogenic diseases by administration oftherapeutic immunological reagents (e.g., humanized immunoglobulins) tospecific epitopes within Aβ to a patient under conditions that generatea beneficial therapeutic response in a patient (e.g., induction ofphagocytosis of Aβ, reduction of plaque burden, inhibition of plaqueformation, reduction of neuritic dystrophy, improving cognitivefunction, and/or reversing, treating or preventing cognitive decline) inthe patient, for example, for the prevention or treatment of anamyloidogenic disease. The invention is also directed to use of thedisclosed immunological reagents (e.g., humanized immunoglobulins) inthe manufacture of a medicament for the treatment or prevention of anamyloidogenic disease.

[0205] The term “treatment” as used herein, is defined as theapplication or administration of a therapeutic agent to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease, a symptom ofdisease or a predisposition toward a disease, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease, the symptoms of disease or the predisposition towarddisease.

[0206] In one aspect, the invention provides methods of preventing ortreating a disease associated with amyloid deposits of Aβ in the brainof a patient. Such diseases include Alzheimer's disease, Down's syndromeand cognitive impairment. The latter can occur with or without othercharacteristics of an amyloidogenic disease. Some methods of theinvention entail administering an effective dosage of an antibody thatspecifically binds to a component of an amyloid deposit to the patient.Such methods are particularly useful for preventing or treatingAlzheimer's disease in human patients. Exemplary methods entailadministering an effective dosage of an antibody that binds to Aβ.Preferred methods entail administering an effective dosage of anantibody that specifically binds to an epitope within residues 1-10 ofAβ, for example, antibodies that specifically bind to an epitope withinresidues 1-3 of Aβ, antibodies that specifically bind to an epitopewithin residues 1-4 of Aβ, antibodies that specifically bind to anepitope within residues 1-5 of Aβ, antibodies that specifically bind toan epitope within residues 1-6 of Aβ, antibodies that specifically bindto an epitope within residues 1-7 of Aβ, or antibodies that specificallybind to an epitope within residues 3-7 of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopecomprising a free N-terminal residue of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopewithin residues of 1-10 of Aβ wherein residue 1 and/or residue 7 of Aβis aspartic acid. In yet another aspect, the invention featuresadministering antibodies that specifically bind to Aβ peptide withoutbinding to full-length amyloid precursor protein (APP). In yet anotheraspect, the isotype of the antibody is human IgG1.

[0207] In yet another aspect, the invention features administeringantibodies that bind to an amyloid deposit in the patient and induce aclearing response against the amyloid deposit. For example, such aclearing response can be effected by Fc receptor mediated phagocytosis.

[0208] Therapeutic agents of the invention are typically substantiallypure from undesired contaminant. This means that an agent is typicallyat least about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

[0209] The methods can be used on both asymptomatic patients and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments) and can bemonoclonal or polyclonal, as described herein. In yet another aspect,the invention features administering antibodies prepared from a humanimmunized with Aβ peptide, which human can be the patient to be treatedwith antibody.

[0210] In another aspect, the invention features administering anantibody with a pharmaceutical carrier as a pharmaceutical composition.Alternatively, the antibody can be administered to a patient byadministering a polynucleotide encoding at least one antibody chain. Thepolynucleotide is expressed to produce the antibody chain in thepatient. Optionally, the polynucleotide encodes heavy and light chainsof the antibody. The polynucleotide is expressed to produce the heavyand light chains in the patient. In exemplary embodiments, the patientis monitored for level of administered antibody in the blood of thepatient.

[0211] The invention thus fulfills a longstanding need for therapeuticregimes for preventing or ameliorating the neuropathology and, in somepatients, the cognitive impairment associated with Alzheimer's disease.

[0212] A. Patients Amenable to Treatment

[0213] Patients amenable to treatment include individuals at risk ofdisease but not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who have a known genetic risk ofAlzheimer's disease. Such individuals include those having relatives whohave experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy,supra). Other markers of risk are mutations in the presenilin genes, PS1and PS2, and ApoE4, family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia, as well as thepresence of risk factors described above. In addition, a number ofdiagnostic tests are available for identifying individuals who have AD.These include measurement of CSF tau and Aβ42 levels. Elevated tau anddecreased Aβ42 levels signify the presence of AD. Individuals sufferingfrom Alzheimer's disease can also be diagnosed by ADRDA criteria asdiscussed in the Examples section.

[0214] In asymptomatic patients, treatment can begin at any age (e.g.,10, 20, 30). Usually, however, it is not necessary to begin treatmentuntil a patient reaches 40, 50, 60 or 70. Treatment typically entailsmultiple dosages over a period of time. Treatment can be monitored byassaying antibody levels over time. If the response falls, a boosterdosage is indicated. In the case of potential Down's syndrome patients,treatment can begin antenatally by administering therapeutic agent tothe mother or shortly after birth.

[0215] B. Treatment Regimes and Dosages In prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of, Alzheimer's disease in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presenting during development ofthe disease. In therapeutic applications, compositions or medicants areadministered to a patient suspected of, or already suffering from such adisease in an amount sufficient to cure, or at least partially arrest,the symptoms of the disease (biochemical, histologic and/or behavioral),including its complications and intermediate pathological phenotypes indevelopment of the disease.

[0216] In some methods, administration of agent reduces or eliminatesmyocognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. The term “immune response”or “immunological response” includes the development of a humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against an antigenin a recipient subject. Such a response can be an active response, i.e.,induced by administration of immunogen, or a passive response, i.e.,induced by administration of immunoglobulin or antibody or primedT-cells.

[0217] An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

[0218] Effective doses of the compositions of the present invention, forthe treatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

[0219] For passive immunization with an antibody, the dosage ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg, preferably at least 1mg/kg. Subjects can be administered such doses daily, on alternativedays, weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

[0220] Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toAβ in the patient. In some methods, dosage is adjusted to achieve aplasma antibody concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, human antibodies show the longesthalf-life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies.

[0221] The dosage and frequency of administration can vary depending onwhether the treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

[0222] In therapeutic applications, a relatively high dosage (e.g., fromabout 1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mgbeing more commonly used) at relatively short intervals is sometimesrequired until progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

[0223] Doses for nucleic acids encoding antibodies range from about 10ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA perpatient. Doses for infectious viral vectors vary from 10-100, or more,virions per dose.

[0224] Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof an immunogenic agent is subcutaneous although other routes can beequally effective. The next most common route is intramuscularinjection. This type of injection is most typically performed in the armor leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionare preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

[0225] Agents of the invention can optionally be administered incombination with other agents that are at least partly effective intreatment of amyloidogenic disease. In the case of Alzheimer's andDown's syndrome, in which amyloid deposits occur in the brain, agents ofthe invention can also be administered in conjunction with other agentsthat increase passage of the agents of the invention across theblood-brain barrier.

[0226] C. Pharmaceutical Compositions

[0227] Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

[0228] Pharmaceutical compositions can also include large, slowlymetabolized macromolecules such as proteins, polysaccharides such aschitosan, polylactic acids, polyglycolic acids and copolymers (such aslatex functionalized sepharose(TM), agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes). Additionally, these carriers can functionas immunostimulating agents (i.e., adjuvants).

[0229] For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

[0230] Typically, compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles such as polylactide, polyglycolide, orcopolymer for enhanced adjuvant effect, as discussed above (see Langer,Science 249: 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97(1997)). The agents of this invention can be administered in the form ofa depot injection or implant preparation, which can be formulated insuch a manner as to permit a sustained or pulsatile release of theactive ingredient.

[0231] Additional formulations suitable for other modes ofadministration include oral, intranasal, and pulmonary formulations,suppositories, and transdermal applications. For suppositories, bindersand carriers include, for example, polyalkylene glycols ortriglycerides; such suppositories can be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

[0232] Topical application can result in transdermal or intradermaldelivery. Topical administration can be facilitated by co-administrationof the agent with cholera toxin or detoxified derivatives or subunitsthereof or other similar bacterial toxins (See Glenn et al., Nature 391,851 (1998)). Co-administration can be achieved by using the componentsas a mixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

[0233] Alternatively, transdermal delivery can be achieved using a skinpath or using transferosomes (Paul et al., Eur. J. Immunol. 25:3521(1995); Cevc et al., Biochem. Biophys. Acta 1368:201-15 (1998)).

[0234] III. Monitoring the Course of Treatment

[0235] The invention provides methods of monitoring treatment in apatient suffering from or susceptible to Alzheimer's, i.e., formonitoring a course of treatment being administered to a patient. Themethods can be used to monitor both therapeutic treatment on symptomaticpatients and prophylactic treatment on asymptomatic patients. Inparticular, the methods are useful for monitoring passive immunization(e.g., measuring level of administered antibody).

[0236] Some methods entail determining a baseline value, for example, ofan antibody level or profile in a patient, before administering a dosageof agent, and comparing this with a value for the profile or level aftertreatment. A significant increase (i.e., greater than the typical marginof experimental error in repeat measurements of the same sample,expressed as one standard deviation from the mean of such measurements)in value of the level or profile signals a positive treatment outcome(i.e., that administration of the agent has achieved a desiredresponse). If the value for immune response does not changesignificantly, or decreases, a negative treatment outcome is indicated.

[0237] In other methods, a control value (i.e., a mean and standarddeviation) of level or profile is determined for a control population.Typically the individuals in the control population have not receivedprior treatment. Measured values of the level or profile in a patientafter administering a therapeutic agent are then compared with thecontrol value. A significant increase relative to the control value(e.g., greater than one standard deviation from the mean) signals apositive or sufficient treatment outcome. A lack of significant increaseor a decrease signals a negative or insufficient treatment outcome.Administration of agent is generally continued while the level isincreasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

[0238] In other methods, a control value of the level or profile (e.g.,a mean and standard deviation) is determined from a control populationof individuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a patient are compared with thecontrol value. If the measured level in a patient is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment may be indicated.

[0239] In other methods, a patient who is not presently receivingtreatment but has undergone a previous course of treatment is monitoredfor antibody levels or profiles to determine whether a resumption oftreatment is required. The measured level or profile in the patient canbe compared with a value previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in

[0240] The tissue sample for analysis is typically blood, plasma, serum,mucous fluid or cerebrospinal fluid from the patient. The sample isanalyzed, for example, for levels or profiles of antibodies to Aβpeptide, e.g., levels or profiles of humanized antibodies. ELISA methodsof detecting antibodies specific to Aβ are described in the Examplessection. In some methods, the level or profile of an administeredantibody is determined using a clearing assay, for example, in an invitro phagocytosis assay, as described herein. In such methods, a tissuesample from a patient being tested is contacted with amyloid deposits(e.g., from a PDAPP mouse) and phagocytic cells bearing Fc receptors.Subsequent clearing of the amyloid deposit is then monitored. Theexistence and extent of clearing response provides an indication of theexistence and level of antibodies effective to clear Aβ in the tissuesample of the patient under test.

[0241] The antibody profile following passive immunization typicallyshows an immediate peak in antibody concentration followed by anexponential decay. Without a further dosage, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example the half-life ofsome human antibodies is of the order of 20 days.

[0242] In some methods, a baseline measurement of antibody to Aβ in thepatient is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

[0243] Additional methods include monitoring, over the course oftreatment, any art-recognized physiologic symptom (e.g., physical ormental symptom) routinely relied on by researchers or physicians todiagnose or monitor amyloidogenic diseases (e.g., Alzheimer's disease).For example, one can monitor cognitive impairment. The latter is asymptom of Alzheimer's disease and Down's syndrome but can also occurwithout other characteristics of either of these diseases. For example,cognitive impairment can be monitored by determining a patient's scoreon the Mini-Mental State Exam in accordance with convention throughoutthe course of treatment.

[0244] C. Kits

[0245] The invention further provides kits for performing the monitoringmethods described above. Typically, such kits contain an agent thatspecifically binds to antibodies to Aβ. The kit can also include alabel. For detection of antibodies to Aβ, the label is typically in theform of labeled anti-idiotypic antibodies. For detection of antibodies,the agent can be supplied prebound to a solid phase, such as to thewells of a microtiter dish. Kits also typically contain labelingproviding directions for use of the kit. The labeling may also include achart or other correspondence regime correlating levels of measuredlabel with levels of antibodies to Aβ. The term labeling refers to anywritten or recorded material that is attached to, or otherwiseaccompanies a kit at any time during its manufacture, transport, sale oruse. For example, the term labeling encompasses advertising leaflets andbrochures, packaging materials, instructions, audio or videocassettes,computer discs, as well as writing imprinted directly on kits.

[0246] The invention also provides diagnostic kits, for example,research, detection and/or diagnostic kits (e.g., for performing in vivoimaging). Such kits typically contain an antibody for binding to anepitope of Aβ, preferably within residues 1-10. Preferably, the antibodyis labeled or a secondary labeling reagent is included in the kit.Preferably, the kit is labeled with instructions for performing theintended application, for example, for performing an in vivo imagingassay. Exemplary antibodies are those described herein.

[0247] D. In Vivo Imaging

[0248] The invention provides methods of in vivo imaging amyloiddeposits in a patient. Such methods are useful to diagnose or confirmdiagnosis of Alzheimer's disease, or susceptibility thereto. Forexample, the methods can be used on a patient presenting with symptomsof dementia. If the patient has abnormal amyloid deposits, then thepatient is likely suffering from Alzheimer's disease. The methods canalso be used on asymptomatic patients. Presence of abnormal deposits ofamyloid indicates susceptibility to future symptomatic disease. Themethods are also useful for monitoring disease progression and/orresponse to treatment in patients who have been previously diagnosedwith Alzheimer's disease.

[0249] The methods work by administering a reagent, such as antibodythat binds to Aβ, to the patient and then detecting the agent after ithas bound. Preferred antibodies bind to Aβ deposits in a patient withoutbinding to full length APP polypeptide. Antibodies binding to an epitopeof Aβ within amino acids 1-10 are particularly preferred. In somemethods, the antibody binds to an epitope within amino acids 7-10 of Aβ.Such antibodies typically bind without inducing a substantial clearingresponse. In other methods, the antibody binds to an epitope withinamino acids 1-7 of Aβ. Such antibodies typically bind and induce aclearing response to Aβ. However, the clearing response can be avoidedby using antibody fragments lacking a full-length constant region, suchas Fabs. In some methods, the same antibody can serve as both atreatment and diagnostic reagent. In general, antibodies binding toepitopes C-terminal to residue 10 of Aβ do not show as strong a signalas antibodies binding to epitopes within residues 1-10, presumablybecause the C-terminal epitopes are inaccessible in amyloid deposits.Accordingly, such antibodies are less preferred.

[0250] Diagnostic reagents can be administered by intravenous injectioninto the body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for Aβ is unlabelled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

[0251] Diagnosis is performed by comparing the number, size, and/orintensity of labeled loci, to corresponding baseline values. The baseline values can represent the mean levels in a population of undiseasedindividuals. Baseline values can also represent previous levelsdetermined in the same patient. For example, baseline values can bedetermined in a patient before beginning treatment, and measured valuesthereafter compared with the baseline values. A decrease in valuesrelative to baseline signals a positive response to treatment.

[0252] The present invention will be more fully described by thefollowing non-limiting examples.

EXAMPLES Example I Therapeutic Efficacy of Anti-Aβ Antibodies: mAb 2H3,mAb 10D5, mAb 266, mAb 21F12 and pAb Aβ1-42

[0253] This example tests the capacity of various monoclonal andpolyclonal antibodies to Aβ to inhibit accumulation of Aβ in the brainof heterozygotic transgenic mice.

[0254] A. Study Design

[0255] Sixty male and female, heterozygous PDAPP transgenic mice, 8.5 to10.5 months of age were obtained from Charles River Laboratory. The micewere sorted into six groups to be treated with various antibodiesdirected to Aβ. Animals were distributed to match the gender, age,parentage and source of the animals within the groups as closely aspossible. Table 2 depicts the Experimental design. TABLE 2 ExperimentalDesign Treatment Treatment Antibody Antibody Group N^(a) AntibodySpecificity Isotype 1 9 none NA^(b) NA (PBS alone) 2 10 PolyclonalAβ1-42 mixed 3 0 mAb^(d)2H3 Aβ1-12 IgG1 4 8 mAb 10D5 Aβ3-7 IgG1 5 6 mAb266 Aβ13-28 IgG1 6 8 mAb 21F12 Aβ33-42 IgG2a

[0256] As shown in Table 2, the antibodies included four murineAβ-specific monoclonal antibodies, 2H3 (directed to Aβ residues 1-12),10D5 (directed to Aβ residues 3-7) (10D5 is produced by the cell linedesignated RB44-10D5.19.21, which was deposited on Apr. 8, 2003 andassigned ATCC accession number PTA-5129), 266 (directed to Aβ residues13-28 and binds to soluble but not to aggregated AN1792), 21F12(directed to Aβ residues 33-42). A fifth group was treated with anAβ-specific polyclonal antibody fraction (raised by immunization withaggregated AN1792). The negative control group received the diluent,PBS, alone without antibody.

[0257] B. Monitoring the Course of Treatment

[0258] The monoclonal antibodies were injected at a dose of about 10mg/kg (assuming that the mice weighed 50 g). Antibody titers weremonitored over the 28 weeks of treatment. Injections were administeredintraperitoneally every seven days on average to maintain anti-Aβ titersabove 1000. Although lower titers were measured for mAb 266 since itdoes not bind well to the aggregated AN1792 used as the capture antigenin the assay, the same dosing schedule was maintained for this group.The group receiving monoclonal antibody 2H3 was discontinued within thefirst three weeks since the antibody was cleared too rapidly in vivo.

[0259] For determination of antibody titers, a subset of three randomlychosen mice from each group were bled just prior to each intraperitonealinoculation, for a total of 30 bleeds. Antibody titers were measured asAβ1-42-binding antibody using a sandwich plastic multi-well platescoated with Aβ1-42 as described in detail in the General Materials andMethods. Mean titers for each bleed are set forth in Table 3 for thepolyclonal antibody and the monoclonals 10D5 and 21F12. TABLE 3 weeksweeks 21F12 21F12 10D5 10D5 weeks poly poly 0.15 500 0.15 3000 0.15 16000.5 800 0.5 14000 0.5 4000 1 2500 1 5000 1 4500 1.5 1800 1.1 5000 1.53000 2 1400 1.2 1300 2 1300 3 6000 2 3000 3 1600 3.5 550 3 4000 3.5 6504 1600 3.5 500 4 1300 5 925 4 2400 5 450 6 3300 5 925 6 2100 7 4000 61700 7 1300 8 1400 7 1600 8 2300 9 1900 8 4000 9 700 10 1700 9 1800 10600 11 1600 10 1800 11 600 12 1000 11 2300 12 1000 13 1500 12 2100 13900 14 1300 13 2800 14 1900 15 1000 14 1900 15 1200 16 1700 15 2700 16700 17 1700 16 1300 17 2100 18 5000 17 2200 18 1800 19 900 18 2200 191800 20 300 19 2500 20 1200 22 1750 20 980 22 1000 23 1600 22 2000 231200 24 1000 23 1000 24 675 25 1100 24 850 25 850 26 2250 25 600 26 160027 1400 26 1100 27 1900 28 27 1450 28 28

[0260] Titers averaged about 1000 over this time period for thepolyclonal antibody preparation and were slightly above this level forthe 10D5- and 21F12-treated animals.

[0261] Treatment was continued over a six-month period for a total of196 days. Animals were euthanized one week after the final dose.

[0262] C. Aβ and APP Levels in the Brain:

[0263] Following about six months of treatment with the various anti-Aβantibody preparations, brains were removed from the animals followingsaline perfusion. One hemisphere was prepared for immunohistochemicalanalysis and the second was used for the quantitation of Aβ and APPlevels. To measure the concentrations of various forms of beta amyloidpeptide and amyloid precursor protein (APP), the hemisphere wasdissected and homogenates of the hippocampal, cortical, and cerebellarregions were prepared in 5M guanidine. These were serially diluted andthe level of amyloid peptide or APP was quantitated by comparison to aseries of dilutions of standards of Aβ peptide or APP of knownconcentrations in an ELISA format.

[0264] The levels of total Aβ and of Aβ1-42 measured by ELISA inhomogenates of the cortex, and the hippocampus and the level of total Aβin the cerebellum are shown in Tables 4, 5, and 6, respectively. Themedian concentration of total Aβ for the control group, inoculated withPBS, was 3.6-fold higher in the hippocampus than in the cortex (medianof 63,389 ng/g hippocampal tissue compared to 17,818 ng/g for thecortex). The median level in the cerebellum of the control group (30.6ng/g tissue) was more than 2,000-fold lower than in the hippocampus.These levels are similar to those previously reported for heterozygousPDAPP transgenic mice of this age (Johnson-Wood et al., supra).

[0265] For the cortex, one treatment group had a median Aβ level,measured as Aβ1-42, which differed significantly from that of thecontrol group (p<0.05), those animals receiving the polyclonal anti-Aβantibody as shown in Table 4. The median level of Aβ1-42 was reduced by65%, compared to the control for this treatment group. The median levelsof Aβ1-42 were also significantly reduced by 55% compared to the controlin one additional treatment group, those animals dosed with the mAb 10D5(p=0.0433). TABLE 4 CORTEX Medians Total Aβ Aβ42 Means Treatment ELISAELISA Total Aβ Aβ42 Group N^(a) value^(b) P value^(c) % Change value Pvalue % Change ELISA value ELISA value PBS 9 17818 NA^(d) NA 13802 NA NA  16150 +/− 7456^(e)  12621 +/− 5738  Polyclonal anti- 10 6160 0.0055−65 4892 0.0071 −65 5912 +/− 4492 4454 +/− 3347 Aβ42 mAb 10D5 8 79150.1019 −56 6214 0.0433 −55 9695 +/− 6929 6943 +/− 3351 mAb 266 6 91440.1255 −49 8481 0.1255 −39 9204 +/− 9293 7489 +/− 6921 mAb 21F12 8 151580.2898 −15 13578 0.7003  −2 12481 +/− 7082  11005 +/− 6324 

[0266] In the hippocampus, the median percent reduction of total Aβassociated with treatment with polyclonal anti-Aβ antibody (50%,p=0.0055) was not as great as that observed in the cortex (65%) (Table5). However, the absolute magnitude of the reduction was almost 3-foldgreater in the hippocampus than in the cortex, a net reduction of 31,683ng/g tissue in the hippocampus versus 11,658 ng/g tissue in the cortex.When measured as the level of the more amyloidogenic form of Aβ, Aβ1-42,rather than as total Aβ, the reduction achieved with the polyclonalantibody was significant (p=0.0025). The median levels in groups treatedwith the mAbs 10D5 and 266 were reduced by 33% and 21%, respectively.TABLE 5 HIPPOCAMPUS Medians Total Aβ Aβ42 Means Treatment ELISA P %ELISA P % Total Aβ Aβ42 Group N^(a) value^(b) value^(c) Change valuevalue Change ELISA value ELISA value PBS 9 63389 NA^(d) NA 54429 NA NA 58351 +/− 13308^(e) 52801 +/− 14701 Polyclonal 10 31706 0.0055 −5027127 0.0025 −50 30058 +/− 22454 24853 +/− 18262 anti-Aβ42 mAb 10D5 846779 0.0675 −26 36290 0.0543 −33 44581 +/− 18632 36465 +/− 17146 mAb266 6 48689 0.0990 −23 43034 0.0990 −21 36419 +/− 27304 32919 +/− 25372mAb 21F12 8 51563 0.7728 −19 47961 0.8099 −12 57327 +/− 28927 50305 +/−23927

[0267] Total Aβ was also measured in the cerebellum (Table 6). Thosegroups dosed with the polyclonal anti-Aβ and the 266 antibody showedsignificant reductions of the levels of total Aβ (43% and 46%, p=0.0033and p=0.0184, respectively) and that group treated with 10D5 had a nearsignificant reduction (29%, p=0.0675). TABLE 6 CEREBELLUM Medians TotalAβ Means Treatment ELISA P % Total Aβ Group N^(a) value^(b) value^(c)Change ELISA value PBS 9 30.64 NA^(d) NA   40.00 +/− 31.89^(e)Polyclonal 10 17.61 0.0033 −43 18.15 +/− 4.36  anti-Aβ42 mAb 10D5 821.68 0.0675 −29 27.29 +/− 19.43 mAb 266 6 16.59 0.0184 −46 19.59 +/−6.59  mAb 21F12 8 29.80 >0.9999  −3 32.88 +/− 9.90 

[0268] APP concentration was also determined by ELISA in the cortex andcerebellum from antibody-treated and control, PBS-treated mice. Twodifferent APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α, the secreted form of APP which has beencleaved within the Aβ sequence), and full-length forms (FL) of APP,while the second recognizes only APP-α. In contrast to thetreatment-associated diminution of Aβ in a subset of treatment groups,the levels of APP were virtually unchanged in all of the treatedcompared to the control animals. These results indicate that theimmunizations with Aβ antibodies deplete Aβ without depleting APP.

[0269] In summary, Aβ levels were significantly reduced in the cortex,hippocampus and cerebellum in animals treated with the polyclonalantibody raised against AN1792. To a lesser extent monoclonal antibodiesto the amino terminal region of Aβ1-42, specifically amino acids 1-16and 13-28 also showed significant treatment effects.

[0270] D. Histochemical Analyses:

[0271] The morphology of Aβ-immunoreactive plaques in subsets of brainsfrom mice in the PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treatmentgroups was qualitatively compared to that of previous studies in whichstandard immunization procedures with Aβ42 were followed.

[0272] The largest alteration in both the extent and appearance ofamyloid plaques occurred in the animals immunized with the polyclonalAβ42 antibody. The reduction of amyloid load, eroded plaque morphologyand cell-associated Aβ immunoreactivity closely resembled effectsproduced by the standard immunization procedure. These observationssupport the ELISA results in which significant reductions in both totalAβ and Aβ42 were achieved by administration of the polyclonal Aβ42antibody.

[0273] In similar qualitative evaluations, amyloid plaques in the 10D5group were also reduced in number and appearance, with some evidence ofcell-associated Aβ immunoreactivity. Relative to control-treatedanimals, the polyclonal Ig fraction against Aβ and one of the monoclonalantibodies (10D5) reduced plaque burden by 93% and 81%, respectively(p<0.005). 21F12 appeared to have a relatively modest effect on plaqueburden. Micrographs of brain after treatment with pAbAβ₁₋₄₂ show diffusedeposits and absence of many of the larger compacted plaques in thepAbAβ₁₋₄₂ treated group relative to control treated animals.

[0274] E. Lymphoproliferative Responses

[0275] Aβ-dependent lymphoproliferation was measured using spleen cellsharvested eight days following the final antibody infusion. Freshlyharvested cells, 10⁵ per well, were cultured for 5 days in the presenceof Aβ1-40 at a concentration of 5 μM for stimulation. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

[0276] Splenocytes from aged PDAPP mice passively immunized with variousanti-Aβ antibodies were stimulated in vitro with AN1792 andproliferative and cytokine responses were measured. The purpose of theseassays was to determine if passive immunization facilitated antigenpresentation, and thus priming of T cell responses specific for AN1792.No AN1792-specific proliferative or cytokine responses were observed inmice passively immunized with the anti-Aβ antibodies.

Example II Therapeutic Efficacy of Anti-Aβ Antibodies: mAb 2H3, mAb10D5, mAb 266, mAb 21F12, mAb 3D6, mAb 16C11 and pAb Aβ1-42

[0277] In a second study, treatment with 10D5 was repeated and twoadditional anti-Aβ antibodies were tested, monoclonals 3D6 (Aβ1-5) and16C11 (Aβ33-42). Control groups received either PBS or an irrelevantisotype-matched antibody (TM2a). The mice were older (11.5-12 month oldheterozygotes) than in the previous study, otherwise the experimentaldesign was the same. Once again, after six months of treatment, 10D5reduced plaque burden by greater than 80% relative to either the PBS orisotype-matched antibody controls (p=0.003). One of the other antibodiesagainst Aβ, 3D6, was equally effective, producing an 86% reduction(p=0.003). In contrast, the third antibody against the peptide, 16C11,failed to have any effect on plaque burden. Similar findings wereobtained with Aβ42 ELISA measurements.

[0278] These results demonstrate that an antibody response against Aβpeptide, in the absence of T cell immunity, is sufficient to decreaseamyloid deposition in PDAPP mice, but that not all anti-Aβ antibodiesare equally efficacious. Antibodies directed to epitopes comprisingamino acids 1-5 or 3-7 of Aβ are particularly efficacious. In summary,it can be demonstrated that passively administered antibodies against Aβ(i.e., passive immunization) reduces the extent of plaque deposition ina mouse model of Alzheimer's disease.

Example III Monitoring of Antibody Binding in the CNS

[0279] This Example demonstrates that when held at modest serumconcentrations (25-70 μg/ml), the antibodies gained access to the CNS atlevels sufficient to decorate β-amyloid plaques.

[0280] To determine whether antibodies against Aβ could be actingdirectly within the CNS, brains taken from saline-perfused mice at theend of the Example II, were examined for the presence of theperipherally-administered antibodies. Unfixed cryostat brain sectionswere exposed to a fluorescent reagent against mouse immunoglobulin (goatanti-mouse IgG-Cy3). Plaques within brains of the 10D5 and 3D6 groupswere strongly decorated with antibody, while there was no staining inthe 16C11 group. To reveal the full extent of plaque deposition, serialsections of each brain were first immunoreacted with an anti-Aβantibody, and then with the secondary reagent. 10D5 and 3D6, followingperipheral administration, gained access to most plaques within the CNS.The plaque burden was greatly reduced in these treatment groups comparedto the 16C11 group. Antibody entry into the CNS was not due to abnormalleakage of the blood-brain barrier since there was no increase invascular permeability as measured by Evans Blue in PDAPP mice. Inaddition, the concentration of antibody in the brain parenchyma of agedPDAPP mice was the same as in non-transgenic mice, representing 0.1% ofthe antibody concentration in serum (regardless of isotype).

[0281] These data indicate that peripherally administered antibodies canenter the CNS where they can directly trigger amyloid clearance. It islikely that 16C11 also had access to the plaques but was unable to bind.

Example IV Ex Vivo Screening Assay for Activity of an Antibody AgainstAmyloid Deposits

[0282] To examine the effect of antibodies on plaque clearance, weestablished an ex vivo assay in which primary microglial cells werecultured with unfixed cryostat sections of either PDAPP mouse or humanAD brains. Microglial cells were obtained from the cerebral cortices ofneonate DBA/2N mice (1-3 days). The cortices were mechanicallydissociated in HBSS^(—)(Hanks' Balanced Salt Solution, Sigma) with 50μg/ml DNase I (Sigma). The dissociated cells were filtered with a 100 μmcell strainer (Falcon), and centrifuged at 1000 rpm for 5 minutes. Thepellet was resuspended in growth medium (high glucose DMEM, 10% FBS, 25ng/ml rmGM-CSF), and the cells were plated at a density of 2 brains perT-75 plastic culture flask. After 7-9 days, the flasks were rotated onan orbital shaker at 200 rpm for 2 h at 37° C. The cell suspension wascentrifuged at 1000 rpm and resuspended in the assay medium.

[0283] 10-μm cryostat sections of PDAPP mouse or human AD brains(post-mortem interval<3 hr) were thaw mounted onto poly-lysine coatedround glass coverslips and placed in wells of 24-well tissue cultureplates. The coverslips were washed twice with assay medium consisting ofH-SFM (Hybridoma-serum free medium, Gibco BRL) with 1% FBS, glutamine,penicillin/streptomycin, and 5 ng/ml rmGM-CSF (R&D). Control or anti-Aβantibodies were added at a 2× concentration (5 μg/ml final) for 1 hour.The microglial cells were then seeded at a density of 0.8×10⁶ cells/mlassay medium. The cultures were maintained in a humidified incubator(37° C., 5% CO₂) for 24 hr or more. At the end of the incubation, thecultures were fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton-X100. The sections were stained with biotinylated 3D6 followed bya streptavidin/Cy3 conjugate (Jackson ImmunoResearch). The exogenousmicroglial cells were visualized by a nuclear stain (DAPI). The cultureswere observed with an inverted fluorescent microscope (Nikon, TE300) andphotomicrographs were taken with a SPOT digital camera using SPOTsoftware (Diagnostic instruments). For Western blot analysis, thecultures were extracted in 8M urea, diluted 1:1 in reducing tricinesample buffer and loaded onto a 16% tricine gel (Novex). After transferonto immobilon, blots were exposed to 5 μg/ml of the pabAβ42 followed byan HRP-conjugated anti-mouse antibody, and developed with ECL (Amersham)

[0284] When the assay was performed with PDAPP brain sections in thepresence of 16C11 (one of the antibodies against Aβ that was notefficacious in vivo), β-amyloid plaques remained intact and nophagocytosis was observed. In contrast, when adjacent sections werecultured in the presence of 10D5, the amyloid deposits were largely goneand the microglial cells showed numerous phagocytic vesicles containingAβ. Identical results were obtained with AD brain sections; 10D5 inducedphagocytosis of AD plaques, while 16C11 was ineffective. In addition,the assay provided comparable results when performed with either mouseor human microglial cells, and with mouse, rabbit, or primate antibodiesagainst Aβ.

[0285] Table 7 compares Aβ binding versus phagocytosis for severaldifferent antibody binding specificities. It can be seen that antibodiesbinding to epitopes within aa 1-7 both bind and clear amyloid deposits,whereas antibodies binding to epitopes within amino acids 4-10 bindwithout clearing amyloid deposits. Antibodies binding to epitopesC-terminal to residue 10 neither bind nor clear amyloid deposits. TABLE7 Analysis of Epitope Specificity Antibody epitope isotype StainingPhagocytosis N-Term mab 3D6 1-5 IgG2b + + 10D5 3-7 IgG1 + + 22C8 3-7IgG2a + + 6E10  5-10 IgG1 + − 14A8  4-10 rat IgG1 + − aa 13-28 18G1110-18 rat IgG1 − − 266 16-24 IgG1 − − 22D12 18-21 IgG2b − − C-Term 2G3−40 IgG1 − − 16C11 −40/−42 IgG1 − − 21F12 −42 IgG2a − − Immune serumrabbit (CFA) 1-6 + + mouse (CFA) 3-7 + + mouse (QS-21) 3-7 + + monkey(QS-21) 1-5 + + mouse (MAP1-7) + +

[0286] Table 8 shows results obtained with several antibodies againstAβ, comparing their abilities to induce phagocytosis in the ex vivoassay and to reduce in vivo plaque burden in passive transfer studies.Although 16C11 and 21F12 bound to aggregated synthetic Aβ peptide withhigh avidity, these antibodies were unable to react with β-amyloidplaques in unfixed brain sections, could not trigger phagocytosis in theex vivo assay, and were not efficacious in vivo. 10D5, 3D6, and thepolyclonal antibody against Aβ were active by all three measures. Theseresults show that efficacy in vivo is due to direct antibody mediatedclearance of the plaques within the CNS, and that the ex vivo assay ispredictive of in vivo efficacy. TABLE 8 The ex vivo assay as predictorof in vivo efficacy Avidity for Binding to aggregated β-amyloid Ex vivoIn vivo Antibody Isotype Aβ (pM) plaques efficacy efficacy monoclonal3D6 IgG2b 470 + + + 10D5 IgG1 43 + + + 16C11 IgG1 90 − − − 21F12 IgG2a500 − − − TM2a IgG1 — − − − polyclonal 1-42 mix 600 + + +

[0287] The same assay has been used to test clearing activity of anantibody against a fragment of synuclein referred to as NAC. Synucleinhas been shown to be an amyloid plaque-associated protein. An antibodyto NAC was contacted with a brain tissue sample containing amyloidplaques, and microglial cells, as before. Rabbit serum was used as acontrol. Subsequent monitoring showed a marked reduction in the numberand size of plaques indicative of clearing activity of the antibody.

[0288] Confocal microscopy was used to confirm that Aβ was internalizedduring the course of the ex vivo assay. In the presence of controlantibodies, the exogenous microglial cells remained in a confocal planeabove the tissue, there were no phagocytic vesicles containing Aβ, andthe plaques remained intact within the section. In the presence of 10D5,nearly all plaque material was contained in vesicles within theexogenous microglial cells. To determine the fate of the internalizedpeptide, 10D5 treated cultures were extracted with 8M urea at varioustime-points, and examined by Western blot analysis. At the one hour timepoint, when no phagocytosis had yet occurred, reaction with a polyclonalantibody against Aβ revealed a strong 4 kD band (corresponding to the Aβpeptide). Aβ immunoreactivity decreased at day 1 and was absent by day3. Thus, antibody-mediated phagocytosis of Aβ leads to its degradation.

[0289] To determine if phagocytosis in the ex vivo assay wasFc-mediated, F(ab′)2 fragments of the anti-Aβ antibody 3D6 wereprepared. Although the F(ab′)2 fragments retained their full ability toreact with plaques, they were unable to trigger phagocytosis bymicroglial cells. In addition, phagocytosis with the whole antibodycould be blocked by a reagent against murine Fc receptors(anti-CD16/32). These data indicate that in vivo clearance of Aβ occursthrough Fc-receptor mediated phagocytosis.

Example V Passage of Antibodies Through the Blood-Brain Barrier

[0290] This example determines the concentration of antibody deliveredto the brain following intravenous injection into a peripheral tissue ofeither normal or PDAPP mice. Following treatment, PDAPP or controlnormal mice were perfused with 0.9% NaCl. Brain regions (hippocampus orcortex) were dissected and rapidly frozen. Brain were homogenized in0.1% triton+protease inhibitors. Immunoglobulin was detected in theextracts by ELISA. F(ab)′2 goat anti-mouse IgG were coated onto an RIAplate as capture reagent. The serum or the brain extracts were incubatedfor 1 hr. The isotypes were detected with anti-mouse IgG1-HRP orIgG2a-HRP or IgG2b-HRP (Caltag). Antibodies, regardless of isotype, werepresent in the CNS at a concentration that is 1:1000 that found in theblood. For example, when the concentration of IgG1 was three times thatof IgG2a in the blood, it was three times IgG2a in the brain as well,both being present at 0.1% of their respective levels in the blood. Thisresult was observed in both transgenic and nontransgenic mice indicatingthat the PDAPP does not have a uniquely leak blood brain barrier.

Example VI Cloning and Sequencing of the Mouse 3D6 Variable Regions

[0291] Cloning and Sequence Analysis of 3D6 VH. The heavy chain variableVH region of 3D6 was cloned by RT-PCR using mRNA prepared from hybridomacells by two independent methods. In the first, consensus primers wereemployed to VH region leader peptide encompassing the translationinitiation codon as the 5′ primer (DNA #3818-3829), and a g2b (DNA#3832) constant regions specific 3′ primer. The sequences from PCRamplified product, as well as from multiple, independently-derivedclones, were in complete agreement with one another. As a further checkon the sequence of the 3D6 VH region, the result was confirmed bysequencing a VH fragment obtained by 5′ RACE RT-PCR methodology and the3′ g2b specific primer (DNA #3832). Again, the sequence was derived fromthe PCR product, as well as multiple, independently-isolated clones.Both sequences are in complete agreement with one another, (with theexception of V8I substitution in the leader region from the 5′ RACEproduct), indicating that the sequences are derived from the mRNAencoding the VH region of 3D6. The nucleotide (SEQ ID NO:3) and aminoacid sequence (SEQ ID NO:4) of the VH region of 3D6 are set forth inTable 9A and in FIG. 2, respectively. TABLE 9A Mouse 3D6 VH NucleotideSequence ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGA(SEQ ID NO:3)AGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGCGTCTCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGGCATGTCTTGGGTTCGCCAGAATTCAGACAAGAGGCTGGAGTGGGTTGCATCCATTAGGAGTGGTGGTGGTAGAACCTACTATTCAGACAATGTAAAGGGCCGATTCACCATCTCCAGAGAGAATGCCAAGAACACCCTGTACCTGCAAATGAGTAGTCTGAAGTCTGAGGACACGGCCTTGTATTATTGTGTCAGATATGATCACTATAGTGGTAGCTCCGACTACTGGGGCCAGGGCACCACT

[0292] Cloning and Sequence Analysis of 3D6 VL. The light chain variableVL region of 3D6 was cloned in an analogous manner as the VH region. Inthe first trial, a consensus primer set was designed for amplificationof murine VL regions as follows: 5′ primers (DNA #3806-3816) weredesigned to hybridize to the VL region encompassing the translationinitiation codon, and a 3′ primer (DNA#3817) was specific for the murineCk region downstream of the V-J joining region. DNA sequence analysis ofthe PCR fragment, as well as independently-derived clones isolated usingthis consensus light chain primer set, revealed that the cDNA obtainedwas derived from a non-functionally rearranged message as the sequencecontained a frameshift mutation between the V-J region junction.

[0293] In a second trial, 5′RACE was employed to clone a second VLencoding cDNA. DNA sequence analysis of this product (consensus 11)showed it encoded a functional mRNA. Thus, it can be concluded that thesequence encodes the correct 3D6 light chain mRNA. The nucleotide (SEQID NO:1) and amino acid sequence (SEQ ID NO:2) of the VL region of 3D6are set forth in Table 9B and in FIG. 1, respectively. TABLE 9B Mouse3D6 VL Nucleotide SequenceATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGG (SEQ IDNO:1) TTATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTTACACTGAAAATCAGCAGAATAGAGGCTGAGGATTTGGGACTTTATTATTGCTGGCAAGGTACACATTTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0294] Primers used for the cloning of the 3D6 VL cDNA are set forth inTABLE 10 Coding DNA Size Strand? DNA Sequence Comments 3806 40 YesACT.AGT.CGA.CAT.GAA.GTT.GCC.TGT.TAG. mouse kappa variableGCT.GTT.GGT.GCT.G (SEQ ID NO:39) primer 1 MKV PRIMER 1, MRC set; % A + T= 50.00 [20]; % C + G = 50.00 [20] Davis, Botstein, Roth Melting Temp C.72.90 3807 39 Yes ACT.AGT.CGA.CAT.GGA.GWC.AGA.CAC.ACT. mouse kappavariable CCT.GYT.ATG.GGT (SEQ ID NO:40) primer 2 MKV PRIMER 2, MRC set %A + T = 46.15 [18]; % C + G = 48.72 [19] Davis, Botstein, Roth MeltingTemp C. 72.05 3808 40 Yes ACT.AGT.CGA.CAT.GAG.TGT.GCT.CAC.TCA. mousekappa variable GGT.CCT.GGS.GTT.G (SEQ ID NO:41) primer 3 MKV PRIMER 3,MRC set; % A + T = 45.00 [18]; % C + G = 52.50 [21] Davis, Botstein,Roth Melting Temp C. 73.93 3809 43 YesACT.AGT.CGA.CAT.GAG.GRC.CCC.TGC.TCA. mouse kappa variableGWT.TYT.TGG.MWT.CTT.G (SEQ ID NO:42) primer 4 MKV PRIMER 4, MRC set; %A + T = 41.86 [18]; % C + G = 46.51 [20] Davis, Botstein, Roth MeltingTemp C. 72.34 3810 40 Yes ACT.AGT.CGA.CAT.GGA.TTT.WCA.GGT.GCA. mousekappa variable GAT.TWT.CAG.CTT.C. (SEQ ID NO:43) primer 5 MKV PRIMER 5,MRC set % A + T = 52.50 [21]; % C + G = 42.50 [17] Davis, Botstein, RothMelting Temp C. 69.83 3811 37 Yes ACT.AGT.CGA.CAT.GAG.GTK.CYY.TGY.TSA.mouse kappa variable GYT.YCT.GRG.G (SEQ ID NO:44) primer 6 MKV PRIMER 6,MRC set; % A + T = 37.84 [14]; % C + G = 40.54 [15] Davis, Botstein,Roth Melting Temp C. 68.01 3812 41 YesACT.AGT.CGA.CAT.GGG.CWT.CAA.GAT.GGA. mouse kappa variableGTC.ACA.KWY.YCW.GG (SEQ ID NO:45) primer 7 MKV PRIMER 7, MRC set; % A +T = 39.02 [16]; % C + G = 46.34 [19] Davis, Botstein, Roth Melting TempC. 71.70 3813 41 Yes ACT.AGT.CGA.CAT.GTG.GGG.AYC.TKT.TTY. mouse kappavariable CMM.TTT.TTC.AAT.TG (SEQ ID NO:46) primer 8 MKV PRIMER 8, MRCset; % A + T = 53.66 [22]; % C + G = 34.15 [14] Davis, Botstein, RothMelting Temp C. 66.70 3814 35 Yes ACT.AGT.CGA.CAT.GGT.RTC.CWC.ASC.TCA.mouse kappa variable GTT.CCT.TG (SEQ ID NO:47) primer 9 MKV PRIMER 9,MRC set. % A + T = 45.71 [16]; % C + G = 45.71 [16] Davis, Botstein,Roth Melting Temp C. 69.36 3815 37 YesACT.AGT.CGA.CAT.GTA.TAT.ATG.TTT.GTT. mouse kappa variable GTC.TAT.TTC.T(SEQ ID NO:48) primer 10 MKV PRIMER 10, MRC set; % A + T = 70.27 [26]; %C + G = 29.73 [11] Davis, Botstein, Roth Melting Temp C. 63.58 3816 38Yes ACT.AGT.CGA.CAT.GGA.AGC.CCC.AGC.TCA. mouse kappa variableGCT.TCT.CTT.CC (SEQ ID NO:49) primer 11 MKV PRIMER 11, MRC set; % A + T= 44.74 [17]; % C + G = 55.26 [21] Davis, Botstein, Roth Melting Temp C.74.40 3817 27 No GGA.TCC.CGG.GTG.GAT.GGT.GGG.AAG.ATG mouse kappa lightchain (SEQ ID NO:50) reverse primer, aa 116-122; Ck constant regionprimer, MRC set + SmaI site; % A + T 47.06 [8]; % C + G = 52.94 [9]Davis, Botstein, Roth Melting Temp C. 57.19 3818 37 YesACT.AGT.CGA.CAT.GAA.ATG.CAG.CTG.GGT. mouse heavy variable CAT.STT.CTT.C(SEQ ID NO:51) primer 1 MHV primer 1, MRC set; 3819 36 YesACT.AGT.CGA.CAT.GGG.ATG.GAG.CTR.TAT. mouse heavy variable CAT.SYT.CTT(SEQ ID NO:52) primer 2 MHV primer 2, MRC set; 3820 37 YesACT.AGT.CGA.CAT.GAA.GWT.GTG.GTT.AAA. mouse heavy variable CTG.GGT.TTT.T(SEQ ID NO:53) primer 3 MHV primer 3, MRC set; 3821 35 YesACT.AGT.CGA.CAT.GRA.CTT.TGG.GYT.CAG. mouse heavy variable CTT.GRT.TT(SEQ ID NO:54) primer 4 MHV primer 4, MRC set; 3822 40 YesACT.AGT.CGA.CAT.GGA.CTC.CAG.GCT.CAA. mouse heavy variableTTT.AGT.TTT.CCT.T (SEQ ID NO:55) primer 5 MHV primer 5, MRC set; 3823 37Yes ACT.AGT.CGA.CAT.GGC.TGT.CYT.RGS.GCT. mouse heavy variableRCT.CTT.CTG.C (SEQ ID NO:56) primer 6 MHV primer 6, MRC set; 3824 36 YesACT.AGT.CGA.CAT.GGR.ATG.GAG.CKG.GRT. mouse heavy variable CTT.TMT.CTT(SEQ ID NO:57) primer 7 MHV primer 7, MRC set; 3825 33 YesACT.AGT.CGA.CAT.GAG.AGT.GCT.GAT.TCT. mouse heavy variable TTT.GTG (SEQID NO:58) primer 8 MHV primer 8, MRC set; 3826 40 YesACT.AGT.CGA.CAT.GGM.TTG.GGT.GTG.GAM. mouse heavy variableCTT.GCT.ATT.CCT.G (SEQ ID NO:59) primer 9 MHV primer 9, MRC set; 3827 37Yes ACT.AGT.CGA.CAT.GGG.CAG.ACT.TAC.ATT. mouse heavy variableCTC.ATT.CCT.G (SEQ ID NO:60) primer 10 MHV primer 10, MRC set; 3828 38Yes ACT.AGT.CGA.CAT.GGA.TTT.TGG.GCT.GAT. mouse heavy variableTTT.TTT.TAT.TG (SEQ ID NO:61) primer 11 MHV primer 11, MRC set; 3829 37Yes ACT.AGT.CGA.CAT.GAT.GGT.GTT.AAG.TCT. mouse heavy variableTCT.GTA.CCT.G (SEQ ID NO:62) primer 12 MHV primer 12, MRC set; 3832 27No GGA.TCC.CGG.GAG.TGG.ATA.GAC.tGA.TGG mouse IgG2b heavy chain (SEQ IDNO:63) reverse primer aa position 119-124, MRC set;

[0295] From N-terminal to C-terminal, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with thenumbering convention of Kabat et al., supra.

[0296] Expression of Chimeric 3D6 Antibody: The variable heavy and lightchain regions were re-engineered to encode splice donor sequencesdownstream of the respective VDJ or VJ junctions, and cloned into themammalian expression vector pCMV-hγ1 for the heavy chain, and pCMV-hκ1for the light chain. These vectors encode human γ1 and Ck constantregions as exonic fragments downstream of the inserted variable regioncassette. Following sequence verification, the heavy chain and lightchain expression vectors were co-transfected into COS cells. Twodifferent heavy chain clones (H2.2 & H3.2) were independentlyco-transfected with 3 different chimeric light chain clones (L3, L4,&L10) to confirm reproducibility of the result. A chimeric 21.6 antibodytransfection was carried out as a positive control for the vectors.Conditioned media was collected 48 hrs post transfection and assayed bywestern blot analysis for antibody production or ELISA for Aβ binding.

[0297] The multiple transfectants all expressed heavy chain+light chaincombinations which are recognized by a goat anti-human IgG (H+L)antibody on a western blot.

[0298] Direct binding of 3D6 and chimeric 3D6 (PK1614) antibodies to Aβwas tested by ELISA analysis. Chimeric 3D6 was found to bind to Aβ withhigh avidity, similar to that demonstrated by 3D6 (FIG. 3A).Furthermore, an ELISA based competitive inhibition assay revealed thatthe chimeric 3D6 and the murine 3D6 antibody competed equally withbiotinylated-3D6 binding to Aβ (FIG. 3B). The chimeric antibodydisplayed binding properties indistinguishable from the 3D6 referencesample. TABLE 11 Conc (μg/ml) 3D6 PK1614 IgG1 0.037 119.3 0.11 118.6118.9 0.33 99.7 71.25 1 98.63 84.53 134.4

[0299] Moreover, both 3D6 and PK1614 were effective at clearing Aβplaques. The ex vivo assay demonstrates that as the concentration ofantibody increases, the amount of Aβ decreases in a similar manner forboth murine and chimeric 3D6 antibodies. Hence, it can be concluded thatthe sequences encode functional 3D6 heavy chain and light chainsrespectively.

Example VII 3D6 Humanization

[0300] Homology/Molecular Modeling. In order to identify key structuralframework residues in the murine 3D6 antibody, a three-dimensional modelwas generated based on the closest murine antibodies for the heavy andlight chains. For this purpose, an antibody designated 1 CR9 was chosenas a template for modeling the 3D6 light chain (PDB ID: 1CR9, Kanyo etal., supra), and an antibody designated 1OPG was chosen as the templatefor modeling the heavy chain. (PDB ID: 1OPG Kodandapani et al., supra).(See also Table 1.) Amino acid sequence alignment of 3D6 with the lightchain and heavy chain of these antibodies revealed that, with theexception of CDR3 of the heavy chain, the 1CR9 and 1OPG antibodies sharesignificant sequence homology with 3D6. In addition, the CDR loops ofthe selected antibodies fall into the same canonical Chothia structuralclasses as do the CDR loops of 3D6, again excepting CDR3 of the heavychain. Therefore, 1CR9 and 1OPG were initially selected as antibodies ofsolved structure for homology modeling of 3D6.

[0301] A first pass homology model of 3D6 variable region based on theantibodies noted above was constructed using the Look & SegMod ModulesGeneMine (v3.5) software package. This software was purchased under aperpetual license from Molecular Applications Group (Palo Alto, Calif.).This software package, authored by Drs. Michael Levitt and Chris Lee,facilitates the process of molecular modeling by automating the stepsinvolved in structural modeling a primary sequence on a template ofknown structure based on sequence homology. Working on a SiliconGraphics IRIS workstation under a UNIX environment, the modeledstructure is automatically refined by a series of energy minimizationsteps to relieve unfavorable atomic contacts and optimize electrostaticand van der Walls interactions.

[0302] A further refined model was built using the modeling capabilityof Quanta®. A query of the PDB database with CDR3 of the heavy chain of3D6 identified 1qkz as most homologous and having the identical numberof residues as 3D6. Hence, CDR3 of the heavy chain of 3D6 was modeledusing the crystal structure of 1qkz as template. The α-carbon backbonetrace of the 3D6 model is shown in FIG. 4. The VH domain is shown as astippled line, and VL domain is shown as a solid line, and CDR loops areindicated in ribbon form.

[0303] Selection of Human Acceptor Antibody Sequences. Suitable humanacceptor antibody sequences were identified by computer comparisons ofthe amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison was performedseparately for the 3D6 heavy and light chains. In particular, variabledomains from human antibodies whose framework sequences exhibited a highdegree of sequence identity with the murine VL and VH framework regionswere identified by query of the Kabat Database using NCBI BLAST(publicly accessible through the National Institutes of Health NCBIinternet server) with the respective murine framework sequences.

[0304] Two candidate sequences were chosen as acceptor sequences basedon the following criteria: (1) homology with the subject sequence; (2)sharing canonical CDR structures with the donor sequence; and (3) notcontaining any rare amino acid residues in the framework regions. Theselected acceptor sequence for VL is Kabat ID Number (KABID) 019230(Genbank Accession No. S40342), and for VH is KABID 045919 (GenbankAccession No. AF115110). First versions of humanized 3D6 antibodyutilize these selected acceptor antibody sequences.

[0305] Substitution of Amino Acid Residues. As noted supra, thehumanized antibodies of the invention comprise variable frameworkregions substantially from a human immunoglobulin (acceptorimmunoglobulin) and complementarity determining regions substantiallyfrom a mouse immunoglobulin (donor immunoglobulin) termed 3D6. Havingidentified the complementarity determining regions of 3D6 andappropriate human acceptor immunoglobulins, the next step was todetermine which, if any, residues from these components to substitute tooptimize the properties of the resulting humanized antibody. Thecriteria described supra were used to select residues for substitution.

[0306]FIGS. 1 and 2 depict alignments of the original murine 3D6 VL andVH, respectively, with the respective version 1 of the humanizedsequence, the corresponding human framework acceptor sequence and,lastly, the human germline V region sequence showing highest homology tothe human framework acceptor sequence. The shaded residues indicate thecanonical (solid fill), vernier (dotted outline), packing (bold), andrare amino acids (bold italics), and are indicated on the figure. Theasterisks indicate residues backmutated to murine residues in the humanacceptor framework sequence, and CDR regions are shown overlined. Asummary of the changes incorporated into version 1 of humanized 3D6 VHand VL is presented in Table 12. TABLE 12 Summary of changes inhumanized 3D6.v1 Changes VL (112 residues) VH (119 residues) Hu->Mu:Framework  4/112  3/119 (1 canon, 1 packing) CDR1  6/16  3/5 CDR2  4/7 7/14 CDR3  5/8  4/10 Hu->Mu 19/112 (17%) 17/119 (14%) Mu->Hu: Framework13/112 14/119 Backmutation notes  1. I2V which is a canonical  4. S49AVernier/beneath the    position.    CDRs.  2. Y36L which is a packing 5. A93V which is a packing    residue and also lies under the    andvernier zone residue    CDRs  6. K94R which is a canonical  3. L46Rwhich is a packing    residue    residue and lies beneath the    CDRsAcceptor notes  7. KABID 019230/Genbank 11. KABID045919/Genbank   Acc#S40342    Acc#AF115110  8. Hu κ  LC subgroup II 12. Hu HC subgroupIII  9. CDRs from same canonical 13. CDRs from same canonical   structural group as donor    structural group as donor    (m3D6)   (m3D6)    L1 = class 4    H1 = class 1    L2 = class 1    H2 = class3   L3 = class1 14. Recognizes capsular 10. Unknown specificity   polysaccharide of Neisseria    meningitidis Acceptor Germline 15. VH3-2316. A3 & A19

[0307] Tables 13 and 14 set forth Kabat numbering keys for the variouslight and heavy chains, respectively. TABLE 13 Key to Kabat Numberingfor Light Chain A19- mouse HUM KABID Germ- KAB # # TYPE 3D6VL 3D6VL019230 line Comment  1 1 FR1 Y Y D D Rare mouse, may contact CDR  2 2 VV I I Canonical/CDR contact  3 3 V V V V  4 4 M M M M  5 5 T T T T  6 6Q Q Q Q  7 7 T S S S  8 8 P P P P  9 9 L L L L  10 10 T S S S  11 11 L LL L  12 12 S P P P  13 13 V V V V  14 14 T T T T  15 15 I P P P  16 16 GG G G  17 17 Q E E E  18 18 P P P P  19 19 A A A A  20 20 S S S S  21 21I I I I  22 22 S S S S  23 23 C C C C  24 24 CDR1 K K R R  25 25 S S S S 26 26 S S S S  27 27 Q Q Q Q  27A 28 S S S S  27B 29 L L L L  27C 30 LL L L  27D 31 D D H H  27E 32 S S S S  28 33 D D N N  29 34 G G G G  3035 K K Y Y  31 36 T T N N  32 37 Y Y Y Y  33 38 L L L L  34 39 N N D D 35 40 FR2 W W W W  36 41 L L Y Y Packing residue  37 42 L L L L  38 43Q Q Q Q  39 44 R K K K  40 45 P P P P  41 46 G G G G  42 47 Q Q Q Q  4348 S S S S  44 49 P P P P  45 50 K Q Q Q  46 51 R R L L Packing residue 47 52 L L L L  48 53 I I I I  49 54 Y Y Y Y  50 55 CDR2 L L L L  51 56V V G G  52 57 S S S S  53 58 K K N N  54 59 L L R R  55 60 D D A A  5661 S S S S  57 62 FR3 G G G G  58 63 V V V V  59 64 P P P P  60 65 D D DD  61 66 R R R R  62 67 F F F F  63 68 T S S S  64 69 G G G G  65 70 S SS S  66 71 G G G G  67 72 S S S S  68 73 G G G G  69 74 T T T T  70 75 DD D D  71 76 F F F F  72 77 T T T T  3 78 L L L L  74 79 K K K K  75 80I I I I  76 81 S S S S  77 82 R R R R  78 83 I V V V  79 84 E E E E  8085 A A A A  81 86 E E E E  82 87 D D D D  83 88 L V V V  84 89 G G G G 85 90 L V V V  86 91 Y Y Y Y  87 92 Y Y Y Y  88 93 C C C C  89 94 CDR3W W M M  90 95 Q Q Q Q  91 96 G G A A  92 97 T T L L  93 98 H H Q Q  9499 F F T T  95 100 P P P P  96 101 R R R  97 102 T T T  98 103 FR4 F F F 99 104 G G G 100 105 G Q Q 101 106 G G G 102 107 T T T 103 108 K K K104 109 L V V 105 110 E E E 106 111 I I I 106A 112 K K K

[0308] TABLE 14 Key to Kabat Numbering for Heavy Chain Mouse VH3-23 3D6HUM KABID Germ- KAB # # TYPE VH 3D6 VH 045919 line Comment  1 1 FR1 E EE E  2 2 V V V V  3 3 K Q Q Q  4 4 L L L L  5 5 V L L L  6 6 E E E E  77 S S S S  8 8 G G G G  9 9 G G G G  10 10 G G G G  11 11 L L L L  12 12V V V V  13 13 K Q Q Q  14 14 P P P P  15 15 G G G G  16 16 A G G G  1717 S S S S  18 18 L L L L  19 19 K R R R  20 20 L L L L  21 21 S S S S 22 22 C C C C  23 23 A A A A  24 24 A A A A  25 25 S S S S  26 26 G G GG  27 27 F F F F  28 28 T T T T  29 29 F F F F  30 30 S S S S  31 31CDR1 N N S S  32 32 Y Y Y Y  33 33 G G A A  34 34 M M V M  35 35 S S S S 36 36 FR2 W W W W  37 37 V V V V  38 38 R R R R  39 39 Q Q Q Q  40 40 NA A A Rare mouse, replace w/Hum  41 41 S P P P  42 42 D G G G Raremouse, replace w/Hum  43 43 K K K K  44 44 R G G G  45 45 L L L L  46 46E E E E  47 47 W W W W  48 48 V V V V  49 49 A A S S CDR contact/veneer 50 50 CDR2 S S A A  51 51 I I I I  52 52 R R S S  52A 53 S S G G  53 54G G S S  54 55 G G G G  55 56 G G G G  56 57 R R S S  57 58 T T T T  5859 Y Y Y Y  59 60 Y Y Y Y  60 61 S S A A  61 62 D D D D  62 63 N N S S 63 64 V V V V  64 65 K K K K  65 66 G G G G  66 67 FR3 R R R R  67 68 FF F F  68 69 T T T T  69 70 I I I I  70 71 S S S S  71 72 R R R R  72 73E D D D  73 74 N N N N  74 75 A A A S  75 76 K K K K  76 77 N N N N  7778 T S S T  78 79 L L L L  79 80 Y Y Y Y  80 81 L L L L  81 82 Q Q Q Q 82 83 M M M M  82A 84 S N N N  82B 85 S S S S  82C 86 L L L L  83 87 KR R R  84 88 S A A A  85 89 E E E E  86 90 D D D D  87 91 T T T T  88 92A A A A  89 93 L L L V  90 94 Y Y Y Y  91 95 Y Y Y Y  92 96 C C C C  9397 V V A A Packing residue, use mouse  94 98 R R K K Canonical, usemouse  95 99 CDR3 Y Y D  96 100 D D N  97 101 H H Y  98 102 Y Y D  99103 S S F 100 104 G G W 100A 105 S S S 100B 106 S S G 100C 107 — — T100D 108 — — F 101 109 D D D 102 110 Y Y Y 103 111 FR4 W W W 104 112 G GG 105 113 Q Q Q 106 114 G G G 107 115 T T T 108 116 T L L 109 117 V V V110 118 T T T 111 119 V V V 112 120 S S S 113 121 S S S

[0309] The humanized antibodies preferably exhibit a specific bindingaffinity for Aβ of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upperlimit of binding affinity of the humanized antibodies for Aβ is within afactor of three, four or five of that of 3D6 (i.e., ˜10 ⁹ M⁻¹). Oftenthe lower limit of binding affinity is also within a factor of three,four or five of that of 3D6.

[0310] Assembly and Expression of Humanized 3D6 VH and VL, Version 1Briefly, for each V region, 4 large single stranded overlappingoligonucleotides were synthesized. In addition, 4 short PCR primers weresynthesized for each V region to further facilitate assembly of theparticular V region. The DNA sequences of the oligonucleotides employedfor this purpose are shown in Table 15. TABLE 15 DNA oligonucleotidesDNA# SIZE Coding? Sequence comments 4060 136 Yes tccgc aagct tgccg ccacchum 3D6 VL-A ATGGA CATGC GCGTG CCCGC CCAGC TGCTG GGCCT GCTGA TGCTG TGGGTGTCCG GCTCC TCCGG CTACG TGGTG ATGAC CCAGT CCCCC CTGTC CCTGC CCGTG ACCCCCGGCG A (SEQ ID NO:17) 4061 131 No CTGGG GGGAC TGGCC GGGCT hum 3D6 VL-BTCTGC AGCAG CCAGT TCAGG TAGGT CTTGC CGTCG GAGTC CAGCA GGGAC TGGGA GGACTTGCAG GAGAT GGAGG CGGGC TCGCC GGGGG TCACG GGCAG GGACA GGGGG G (SEQ IDNO:18) 4062 146 Yes ACCTG AACTG GCTGC TGCAG hum 3D6 VL-C AAGCC CGGCCAGTCC CCCCA GCGCC TGATC TACCT GGTGT CCAAG CTGGA CTCCG GCGTG CCCGA CCGCTTCTCC GGCTC CGGCT CCGGC ACCGA CTTCA CCCTG AAGAT CTCCC GCGTG GAGGC C (SEQID NO:19) 4063 142 No aattc tagga tccac tcacg hum 3D6 VL-D CTTGA TCTCCACCTT GGTGC CCTGG CCGAA GGTGC GGGGG AAGTG GGTGC CCTGC CAGCA GTAGT ACACGCCCAC GTCCT CGGCC TCCAC GCGGG AGATC TTCAG GGTGA AGTCG GTGCC GG (SEQ IDNO:20) 4064 16 No CTGGG GGGAC TGGCC G hum 3D6 VL A + B (SEQ ID NO:21)back % A + T = 18.75 [3]; % C + G = 81.2 [13] Davis, Botstein, RothMelting Temp C. 66.96 4065 22 Yes ACCTG AACTG GCTGC TGCAG hum 3D6 VL C +D AA (SEQ ID NO:22) forward % A + T = 45.45 [10]; % C + G = 54.55 [12]Davis, Botstein, Roth Melting Temp C. 64.54 4066 138 Yes acaga aagcttgccg ccacc hum 3D6 VH-A ATGGA GTTTG GGCTG AGCTG GCTTT TTCTT GTGGC TATTTTAAAA GGTGT CCAGT GTGAG GTGCA GCTGC TGGAG TCCGG CGGCG GCCTG GTGCA GCCCGGCGGC TCCCT GCGCC TGT (SEQ ID NO:23) 4067 135 No GCCGC CGGAG CGGAT GGAGGhum 3D6 VH-B CCACC CACTC CAGGC CCTTG CCGGG GGCCT GGCGC ACCCA GGACA TGCCGTAGTT GGAGA AGGTG AAGCC GGAGG CGGCG CAGGA CAGGC GCAGG GAGCC GCCGG GCTGCACCAG (SEQ ID NO:24) 4068 142 Yes CTGGA GTGGG TGGCC TCCAT hum 3D6 VH-CCCGCT CCGGC GGCGG CCGCA CCTAC TACTC CGACA ACGTG AAGGG CCGCT TCACC ATCTCCCGCG ACAAC GCCAA GAACT CCCTG TACCT GCAGA TGAAC TCCCT GCGCG CCGAG GACACCG (SEQ ID NO:25) 4069 144 No ctgca aggat ccact caccG hum 3D6 VH-D GAGGACACGG TCACC AGGGT GCCCT GGCCC CAGTA GTCGG AGGAG CCGGA GTAGT GGTCG TAGCGCACGC AGTAG TACAG GGCGG TGTCC TCGGC GCGCA GGGAG TTCAT CTGCA GGTAC AGGG(SEQ ID NO:26) 4070 16 No GCCGC CGGAG CGGAT G hum 3D6 VH A + B (SEQ IDNO:27) back % A + T = 18.75 [3]; % C + G = 81.25 [13] Davis, Botstein,Roth Melting Temp C. 66.96 4071 20 Yes CTGGA GTGGG TGGCC TCCAT hum 3D6VH C + D (SEQ ID NO:28) forward % A + T = 35.00 [7]; % C + G = 65.00[13] Davis, Botstein, Roth Melting Temp C. 66.55 4072 19 Yes tcc gca agcttg ccg cca Hum 3D6 VL A + B c (SEQ ID NO:29) Forward % A + T = 31.58[6]; % C + G = 68.42 [13] Davis, Botstein, Roth Melting Temp C. 66.644073 29 No aat tct agg atc cac tca Hum 3D6 VL C + D cgC TTG ATC TC Back(SEQ ID NO:30) % A + T = 55.17 [16]; % C + G = 44.83 [13] Davis,Botstein, Roth Melting Temp C. 66.04 4074 23 Yes aca gaa agc ttg cog ccaHum 3D6 VH A + B ccA TG Forward (SEQ ID NO:31) % A + T = 43.48 [10]; %C + G = 56.52 [13] Davis, Botstein, Roth Melting Temp C. 66.33 4075 22No ctg caa gga tcc act cac Hum 3D6 VH C + D cGG A Back (SEQ ID NO:32) %A + T = 40.91 [9]; % C + G = 59.09 [13] Davis, Botstein, Roth MeltingTemp C. 66.40

[0311] The humanized light chain was assembled using PCR. DNA sequenceanalysis of greater than two dozen clones revealed scattered pointmutations and deletions throughout the VL region with respect to theexpected sequence. Analysis of the sequences indicated that clone 2.3was amenable to repair of 2 closely spaced single nucleotide deletionsin the amino-terminal region. Hence site directed mutagenesis wasperformed on clone pCRShum3D6v12.3 using oligonucleotides to introducethe 2 deleted nucleotides, and repair of the point mutations wasconfirmed by DNA sequence analysis, and the VL insert was cloned intothe light chain expression vector pCMV-cK.

[0312] Assembly of humanized VH using PCR-based methods resulted inclones with gross deletions in the 5′ half of the sequence. Furtherefforts to optimize the PCR conditions met with partial success. Theclones assembled via optimized PCR conditions still had 10-20 ntdeletions in the region mapping to the overlap of the A+B fragments.Consequently, an alternate strategy was employed for VH assemblyutilizing DNA polymerase (T4, Klenow, and Sequenase) mediated overlapextension, followed by T4 DNA ligase to covalently join the overlappingends. DNA sequence analysis of a subset of the clones resulting from VHassembly using the latter approach revealed scattered point mutationsand deletions among the clones. Analysis of over two dozen clonesrevealed essentially the same pattern as illustrated for the clones. Thesimilar results observed following first pass assembly of VH and VLclones suggests the DNA sequence errors observed resulted from automatedsynthesizer errors during the synthesis of the long DNAs employed forthe assembly.

[0313] Humanized VH clone 2.7 was selected for site-directedmutagenesis-mediated repair of the 3 nucleotide deletions it wasobserved to contain.

Example XIII Characterization of Humanized 3D6v2 Antibody

[0314] A second version of humanized 3D6 was created having each of thesubstitutions indicated for version 1, except for the D→Y substitutionat residue 1. Substitution at this residue was performed in version 1because the residue was identified as a CDR interacting residue.However, substitution deleted a residue which was rare for humanimmunoglobulins at that position. Hence, a version was created withoutthe substitution. Moreover, non-germline residues in the heavy chainframework regions were substituted with germline residues, namely,H74=S, H77=T and H89=V. Kabat numbering for the version 2 light andheavy chains, is the same as that depicted in Tables 13 and 14,respectively, except that residue 1 of the version 2 light chain is asp(D), residue 74 of the heavy chain is ser (S), residue 77 of the heavychain is thr (T) and residue 89 of the heavy chain is val (V). Thenucleotide sequence of humanized 3D6 version 1 light and heavy chainsare set forth as SEQ ID NOs: 34 and 36, respectively. The nucleotidesequence of humanized 3D6 version 2 light and heavy chains are set forthas SEQ ID NOs: 35 and 37, respectively.

Example IX Functional Testing of Humanized 3D6 Antibodies

[0315] Binding of humanized 3D6v1 to aggregated Aβ. Functional testingof humanized 3D6v1 was conducted using conditioned media fromtransiently transfected COS cells. The cells were transfected with fullychimeric antibody, a mixture of either chimeric heavy chain+humanizedlight chain, or chimeric light chain+humanized heavy chain, and lastly,fully humanized antibody. The conditioned media was tested for bindingto aggregated Aβ1-42 by ELISA assay. The humanized antibody showed goodactivity within experimental error, and displayed binding propertiesindistinguishable from the chimeric 3D6 reference sample. The resultsare shown in Table 16. TABLE 16 hu VH/ ChVH/ Hu VH/ ng/ml Chimeric ChVLHuVL HuVL 690 0.867 600 0.895 260 0.83 230 0.774 200 0.81 190 0.811 870.675 77 0.594 67 0.689 63 0.648 29 0.45 25 0.381 22 0.496 21 0.438 9.60.251 8.5 0.198 7.4 0.278 7 0.232 3.2 0.129 2.3 0.124

[0316] To compare the binding affinities of humanized 3D6v1 and 3D6v2antibodies, ELISA analysis was performed using aggregated Aβ as theantigen. The results show that both 3D6v1 (H1L1) and 3D6v2 (H2L2) havenearly identical Aβ binding properties (FIG. 5).

[0317] Replacement NET (rNET) analysis of h3D6v2. The rNET epitope mapassay provides information about the contribution of individual residueswithin the epitope to the overall binding activity of the antibody. rNETanalysis uses synthesized systematic single substituted peptide analogs.Binding of an antibody being tested is determined against native peptide(native antigen) and against 19 alternative “single substituted”peptides, each peptide being substituted at a first position with one of19 non-native amino acids for that position. A profile is generatedreflecting the effect of substitution at that position with the variousnon-native residues. Profiles are likewise generated at successivepositions along the antigenic peptide. The combined profile, or epitopemap, (reflecting substitution at each position with all 19 non-nativeresidues) can then be compared to a map similarly generated for a secondantibody. Substantially similar or identical maps indicate thatantibodies being compared have the same or similar epitope specificity.

[0318] This analysis was performed for 3D6 and humanized 3D6, version 2.Antibodies were tested for binding against the native Aβ peptideDAEFRHDSGY (SEQ ID NO:33). Residues 1-8 were systematically substitutedwith each of the 19 non-native residues for that position. Maps weregenerated accordingly for 3D6 and h3D6v2. The results are presented intabular form in Table 17. TABLE 17 Aβ: replacement Net Epitope (rNET)mapping of wt3D6 and humanized 3D6 Wildtype Humanized 3D6 3D6Substitution [OD] [OD] Residue 1 = A 0.464 0.643 C 0.450 0.628 D 0.5770.692 E 0.576 0.700 F 0.034 0.062 G 0.569 0.738 H 0.054 0.117 I 0.0480.118 K 0.033 0.057 L 0.073 0.148 M 0.039 0.072 N 0.587 0.757 P 0.0690.144 Q 0.441 0.689 R 0.056 0.155 S 0.569 0.762 T 0.450 0.702 V 0.0570.190 W 0.031 0.070 Y 0.341 0.498 Residue 2 = A 0.548 0.698 C 0.5530.694 D 0.119 0.222 E 0.563 0.702 F 0.577 0.717 G 0.527 0.720 H 0.5340.741 I 0.522 0.722 K 0.548 0.722 L 0.482 0.705 M 0.535 0.705 N 0.5250.735 P 0.445 0.707 Q 0.567 0.756 R 0.562 0.719 S 0.587 0.705 T 0.5520.712 V 0.550 0.702 W 0.553 0.701 Y 0.547 0.704 Residue 3 = A 0.0380.061 C 0.222 0.410 D 0.019 0.027 E 0.542 0.689 F 0.034 0.060 G 0.0160.019 H 0.016 0.020 I 0.019 0.024 K 0.053 0.090 L 0.019 0.026 M 0.0190.027 N 0.024 0.032 P 0.017 0.020 Q 0.153 0.406 R 0.015 0.023 S 0.0160.021 T 0.015 0.019 V 0.016 0.021 W 0.149 0.304 Y 0.016 0.020 Residue 4= A 0.016 0.020 C 0.020 0.023 D 0.017 0.020 E 0.016 0.021 F 0.557 0.703G 0.016 0.020 H 0.470 0.723 I 0.119 0.360 K 0.015 0.018 L 0.559 0.716 M0.549 0.725 N 0.085 0.089 P 0.030 0.056 Q 0.065 0.110 R 0.016 0.019 S0.026 0.031 T 0.016 0.021 V 0.213 0.494 W 0.291 0.568 Y 0.529 0.730Residue 5 = A 0.275 0.435 C 0.359 0.635 D 0.080 0.163 E 0.115 0.187 F0.439 0.569 G 0.485 0.679 H 0.577 0.680 I 0.510 0.671 K 0.573 0.693 L0.517 0.691 M 0.418 0.611 N 0.476 0.655 P 0.093 0.198 Q 0.388 0.565 R0.613 0.702 S 0.487 0.633 T 0.530 0.639 V 0.493 0.562 W 0.393 0.461 Y0.278 0.230 Residue 6 = A 0.587 0.707 C 0.585 0.703 D 0.584 0.701 E0.579 0.702 F 0.586 0.704 G 0.592 0.709 H 0.596 0.688 I 0.602 0.708 K0.585 0.691 L 0.584 0.688 M 0.583 0.687 N 0.580 0.686 P 0.587 0.705 Q0.570 0.695 R 0.576 0.686 S 0.573 0.689 T 0.573 0.700 V 0.588 0.715 W0.576 0.696 Y 0.595 0.708 Residue 7 = A 0.580 0.688 C 0.559 0.676 D0.573 0.681 E 0.565 0.677 F 0.546 0.668 G 0.562 0.679 H 0.557 0.675 I0.552 0.681 K 0.565 0.685 L 0.566 0.701 M 0.562 0.697 N 0.573 0.688 P0.582 0.678 Q 0.563 0.679 R 0.551 0.677 S 0.563 0.674 T 0.560 0.685 V0.563 0.687 W 0.547 0.685 Y 0.560 0.682 Residue 8 = A 0.573 0.687 C0.583 0.700 D 0.586 0.697 E 0.601 0.701 F 0.586 0.687 G 0.569 0.681 H0.559 0.683 I 0.568 0.686 K 0.557 0.698 L 0.570 0.686 M 0.571 0.693 N0.573 0.700 P 0.574 0.694 Q 0.590 0.703 R 0.589 0.699 S 0.599 0.719 T0.586 0.689 V 0.578 0.688 W 0.567 0.687 Y 0.574 0.680

[0319] Notably, the profiles are virtually identical for 3D6 and h3D6v2when one looks at the substitutions at each position (i.e., the valuesfluctuate in an identical manner when comparing the data in column 1(3D6) versus column 2 (h3D6v2). These data demonstrate that thespecificity of h3D6v2 is preserved, as the h3D6v2 rNET epitope map isvirtually identical to m3D6 using both Aβ residues 1-4 and 5-8.

[0320] Immunohistochemistry on PDAPP brain sections demonstratesspecificity of h3D6v1 antibody. Humanized 3D6v1 antibody recognized Aβin cryostat prepared brain sections from PDAPP mice. Humanized 3D6v1 andPK1614 both bound to PDAPP plaques in the same dose response fashion, asmeasured by the amount of fluorescence (quantitated in pixels) per slideversus the amount of antibody used to stain the tissue (FIG. 6).Identical anti-human secondary antibodies were used in this experiment.Sectioning, staining, and image procedures were previously described. Inidentical experiments, image analysis of h3D6v2 staining on PDAPP and ADbrain sections revealed that h3D6v2 recognizes Aβ plaques in a similarmanner to 3D6v1 (e.g., highly decorated plaques).

[0321] Competitive binding analysis of h3D6. The ability of h3D6antibodies v1 and v2 to compete with murine 3D6 was measured by ELISAusing a biotinylated 3D6 antibody. Competitive binding analysis revealedthat h3D6v1, h3D6v2, and chimeric PK1614 can all compete with m3D6 tobind Aβ (FIG. 7). h3D6v1 and h3D6v2 were identical in their ability tocompete with 3D6 to AP. The 10D5 antibody was used as a negativecontrol, as it has a different binding epitope than 3D6. BIAcoreanalysis also revealed a high affinity of h3D6v1 and h3D6v2 for Aβ(Table 18). TABLE 18 Affinity Measurements of Aβ Antibodies UsingBIAcore Technology Antibody ka1 (1/Ms) kd1 (1/s) Kd (nM) Mu 3D6 4.06E+053.57E−04 0.88 Chimeric 3D6 4.58E+05 3.86E−04 0.84 Hu 3D6v1 1.85E+053.82E−04 2.06 Hu 3D6v2 1.70E+05 3.78E−04 2.24

[0322] In comparison to 3D6, which has a Kd of 0.88 nM, both h3D6v1 andh3D6v2 had about a 2 to 3 fold less binding affinity, measured at 2.06nM and 2.24 nM for h3D6v1 and h3D6v2, respectively. The ELISAcompetitive binding assay revealed an approximate 6-fold less bindingaffinity for h3D6v1 and h3D6v2. Typically humanized antibodies loseabout 3-4 fold in binding affinity in comparison to their murinecounterparts. Therefore, a loss of about 3 fold (average of ELISA andBIAcore results) for h3D6v1 and h3D6v2 is within the accepted range.

[0323] Ex vivo assay using h3D6v2 antibody. The ability of h3D6v2 tostimulate microglial cells was tested through an ex vivo phagocytosisassay (FIG. 8). h3D6v2 was as effective as chimeric 3D6 at inducingphagocytosis of Aβ aggregates from PDAPP mouse brain tissue. IgG wasused as a negative control in this experiment because it is incapable ofbinding Aβ and therefore cannot induce phagocytosis.

[0324] In vivo brain localization of h3D6. ¹²⁵I labeled h3D6v2, m3D6,and antibody DAE13 were each IV-injected into 14 individual PDAPP micein separate experiments. Mice were sacrificed after Day 7 and perfusedfor further analysis. Their brain regions were dissected and measuredfor ¹²⁵I activity in specific brain regions. Radiolabel activity in thebrain was compared with activity in serum samples. Results are set forthin Tables 19 and 20, for serum and brain regions, respectively. TABLE 19m3D6 DAE13 Hu3D6 30389.1 17463.9 40963.8 12171 13200.6 24202.2 3418.236284.7 12472.4 18678.9 421.3 33851.8 27241 19702 27187.3 26398.824855.8 29016.9 27924.8 29287.4 33830.7 12008.4 12733.1 26734.9 29487.827722.5 30144.5 25498.6 30460.7 35126.9 9652 23320.1 28414.8 24599.37119.1 16956.1 29240 28093.5 18190.7 11922.7 24659.7 25671.4 17443.126748.9

[0325] TABLE 20 m3D6 DAE13 Hu3D6 (H2L2) cere cort hipp cere cort hippcere cort hipp 1991.9 1201.1 4024 1277.5 2522.9 5711.9 2424.6 3759.411622 238.9 746.1 2523 502.5 2123.5 6965.8 1509.8 2274.9 7018.2 645.9603 1241.1 2325 3528.2 7801.6 500 2265.9 5316.3 1000 2508.2 4644.2 232.7849.8 1891.9 2736.2 5703.7 10395.5 1266.9 3737.9 7975.8 891.6 26218245.2 1192.2 3188 10170 1422 2398.7 7731.1 1102.6 2087.5 7292.3 2269.43481.4 9621.6 1700.4 2154.4 7124.1 1650.6 3488.4 10284.8 1526.7 30288331.3 542.5 812.4 2456.8 712.9 2318.5 6643.3 1538.1 4194.1 11244.8 13093010.5 8693.5 1172.9 1953.6 7363 1245.7 1699.4 6831.2 1372.2 997.52425.4 1067.9 3697.2 12280.7 2708.8 2789 7887.4 778.6 1291.9 5654.41952.2 2120.7 6412.7 2251.3 3897.5 11121.5 1199.3 1683.4 4887.3 1005.21852.5 5121.4 1529.6 1772.2 7986.9 1021.8 3234.5 8036.2 961.5 3382.98473.1 644.1 1663.4 5056.5 742.1 1056.7 3405.2 852.3 1943.2 6717.41516.4 1620.6 9888 1273.7 1320.8 4262.6 997.5 3065.7 10213.1

[0326] The data show that h3D6v2 localized to the brain, and wasparticularly concentrated in the hippocampal region where Aβ is known toaggregate. Brain counts for m3D6 and DAE13 were comparable to h3D6v2.All three antibodies were able to cross the blood barrier asdemonstrated by Aβ plaque binding in vivo.

Example X Cloning and Sequencing of the Mouse 10D5 Variable Regions

[0327] Cloning and Sequence Analysis of 10D5 VH. The VH and VL regionsof 10D5 from hybridoma cells were cloned by RT-PCR using 5′ RACEprocedures. The nucleotide sequence (SEQ ID NO:13) and deduced aminoacid sequence (SEQ ID NO:14) derived from two independent cDNA clonesencoding the presumed 10D5 VL domain, are set forth in Table 21 and FIG.9. The nucleotide sequence (SEQ ID NO:15) and deduced amino acidsequence (SEQ ID NO:16) derived from two independent cDNA clonesencoding the presumed 10D5 VH domain, are set forth in Table 22 and FIG.10. The 10D5 VL and VH sequences meet the criteria for functional Vregions in so far as they contain a contiguous ORF from the initiatormethionine to the C-region, and share conserved residues characteristicof immunoglobulin V region genes. TABLE 21 Mouse 10D5 VL DNA sequenceATGAAGTTGCCTGTTAGGCTGTTGGTACTGATGTTCTGGATTCCTGCTTCCAGCAGTGA (SEQ IDNO:13) TGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTATACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAAGAAAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGGAA

[0328] TABLE 22 Mouse 10D5 VH DNA sequence.ATGGACAGGCTTACTTCCTCATTCCTGCTGCTGATTGTCCCTGCATATGTCCTGTCCCA (SEQ IDNO:15) GGCTACTCTGAAAGAGTCTGGCCCTGGAATATTGCAGTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGAGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAAGCAGGTATTCCTCAAGATCACCAGTGTGGACCCTGCAGATACTGCCACATACTACTGTGTTCGAAGGCCCATTACTCCGGTACTAGTCGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

Example XI Efficacy of mAb 3D6 on Various Neuropathological Endpoints inPDAPP Mice

[0329] This Example describes the efficacy of murine mAb 3D6 on variousneuropathological endpoints. A comparison is made between passiveimmunization with 3D6 (at varying doses) and active immunization with anAβ peptide.

[0330] Immunizations

[0331] PDAPP mice were passively immunized with mAb 3D6 at threedifferent doses, 10 mg/kg, 1 mg/kg and 10 mg/kg once a month (1×4). Anunrelated IgGγ2a antibody (TY 11/15) and PBS injections served ascontrols. Active immunization with Aβ peptide served as a comparison.Between 20 and 35 animals were analyzed in each group.

[0332] The neuropathological endpoints assayed include amyloid burdenand neuritic burden.

[0333] Amyloid Burden

[0334] The extent of the frontal cortex occupied by amyloid deposits wasdetermined by immunostaining with 3D6 followed by quantitative imageanalysis. The results of this analysis are shown in Table 6. Each of theimmunotherapies led to a significant reduction of amyloid burden.

[0335] Neuritic Burden

[0336] Neuritic burden following passive immunization with 3D6 wasdetermined in PDAPP mice by immunostaining of brain sections withanti-APP antibody 8E5 followed by quantitative image analysis. Neuriticdystrophy is indicated by the appearance of dystrophic neurites (e.g.,neurites with a globular appearance) located in the immediate vicinityof amyloid plaques. The results of this analysis are shown in Table 7.3D6 (IgGγ2a isotype, recognizing Aβ 1-5) did not significantly reduceneuritic burden as compared to active immunization with Aβ peptide.Previously, it had been observed that 10D5 (IgGγ1 isotype recognizing Aβ3-7) was unable to significantly reduce neuritic burden. TABLE 23Frontal Cortex Amyloid Burden PBS TY 11/15 3D6, 10 mg/kg 3D6, 1 mg/kg3D6, 10 mg/kg/4 wks. Active N 31 30 29 31 32 24 Median (%AB) 15.18229713.303288 0.865671 2.286513 1.470956 2.162772 Range 0.160-31.9610-61.706 0-7.064 0.077-63.362 0-10.688 0-30.715 pVaIue .9425 ns ***.0001***<.0001 ***<.0001 ***.0004 (*M-W) % Change N/A 12% 94% 85% 90% 86%

[0337] TABLE 24 Frontal Cortex Neuritic Burden PBS TY 11/15 3D6, 10mg/kg 3D6, 1 mg/kg 3D6, 10 mg/kg/4 wks. Active N 31 30 29 31 32 24Median (% NB) 0.3946 0.3958 0.4681 0.3649 0.4228 0.2344 Range 0-1.38280-2.6800 0-1.3098 0-1.5760 0-1.8215 0-1.1942 p Value (*M-W) .8967 ns.9587 ns .6986 ns >.9999 ***.0381 % Change 0% 0% 7% 0% 41%

[0338] The characterization of various neuropathological endpoints inthe PDAPP mouse model of Alzheimer's disease may assist the skilledartisan in designing appropriate human therapeutic immunizationprotocols.

Example XII Prevention and Treatment of Human Subjects

[0339] A single-dose phase I trial is performed to determine safety inhumans. A therapeutic agent is administered in increasing dosages todifferent patients starting from about 0.01 the level of presumedefficacy, and increasing by a factor of three until a level of about 10times the effective mouse dosage is reached.

[0340] A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

[0341] Following baseline measures, patients begin receiving treatment.They are randomized and treated with either therapeutic agent or placeboin a blinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

[0342] A second phase II trial is performed to evaluate conversion ofpatients from non-Alzheimer's Disease early memory loss, sometimesreferred to as age-associated memory impairment (AAMI) or mild cognitiveimpairment (MCI), to probable Alzheimer's disease as defined as by ADRDAcriteria. Patients with high risk for conversion to Alzheimer's Diseaseare selected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

[0343] General Materials and Methods

[0344] A. Preparation of Polyclonal and Monoclonal Aβ Antibodies

[0345] The anti-Aβ polyclonal antibody was prepared from blood collectedfrom two groups of animals. The first group consisted of 100 femaleSwiss Webster mice, 6 to 8 weeks of age. They were immunized on days 0,15, and 29 with 100 μg of AN1792 combined with CFA/IFA. A fourthinjection was given on day 36 with one-half the dose of AN 1792. Animalswere exsanguinated upon sacrifice at day 42, serum was prepared and thesera were pooled to create a total of 64 ml. The second group consistedof 24 female mice isogenic with the PDAPP mice but nontransgenic for thehuman APP gene, 6 to 9 weeks of age. They were immunized on days 0, 14,28 and 56 with 100 μg of AN1792 combined with CFA/IFA. These animalswere also exsanguinated upon sacrifice at day 63, serum was prepared andpooled for a total of 14 ml. The two lots of sera were pooled. Theantibody fraction was purified using two sequential rounds ofprecipitation with 50% saturated ammonium sulfate. The final precipitatewas dialyzed against PBS and tested for endotoxin. The level ofendotoxin was less than 1 EU/mg.

[0346] The anti-Aβ monoclonal antibodies were prepared from ascitesfluid. The fluid was first delipidated by the addition of concentratedsodium dextran sulfate to ice-cold ascites fluid by stirring on ice to areach a final concentration of 0.238%. Concentrated CaCl₂ was then addedwith stirring to reach a final concentration of 64 mM. This solution wascentrifuged at 10,000×g and the pellet was discarded. The supernatantwas stirred on ice with an equal volume of saturated ammonium sulfateadded dropwise. The solution was centrifuged again at 10,000×g and thesupernatant was discarded. The pellet was resuspended and dialyzedagainst 20 mM Tris-HCl, 0.4 M NaCl, pH 7.5. This fraction was applied toa Pharmacia FPLC Sepharose Q Column and eluted with a reverse gradientfrom 0.4 M to 0.275 M NaCl in 20 mM Tris-HCl, pH 7.5.

[0347] The antibody peak was identified by absorbance at 280 nm andappropriate fractions were pooled. The purified antibody preparation wascharacterized by measuring the protein concentration using the BCAmethod and the purity using SDS-PAGE. The pool was also tested forendotoxin. The level of endotoxin was less than 1 EU/mg titers, titersless than 100 were arbitrarily assigned a titer value of 25.

[0348] B. Measurement of Antibody Titers

[0349] Mice were bled by making a small nick in the tail vein andcollecting about 200 μl of blood into a microfuge tube. Guinea pigs werebled by first shaving the back hock area and then using an 18 gaugeneedle to nick the metatarsal vein and collecting the blood intomicrofuge tubes. Blood was allowed to clot for one hr at roomtemperature (RT), vortexed, then centrifuged at 14,000×g for 10 min toseparate the clot from the serum. Serum was then transferred to a cleanmicrofuge tube and stored at 4° C. until titered.

[0350] Antibody titers were measured by ELISA. 96-well microtiter plates(Costar EIA plates) were coated with 100 μl of a solution containingeither 10 μg/ml either Aβ42 or SAPP or other antigens as noted in eachof the individual reports in Well Coating Buffer (0.1 M sodiumphosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. Thewells were aspirated and sera were added to the wells starting at a{fraction (1/100)} dilution in Specimen Diluent (0.014 M sodiumphosphate, pH 7.4, 0.15 M NaCl, 0.6% bovine serum albumin, 0.05%thimerosal). Seven serial dilutions of the samples were made directly inthe plates in three-fold steps to reach a final dilution of {fraction(1/218,700)}. The dilutions were incubated in the coated-plate wells forone hr at RT. The plates were then washed four times with PBS containing0.05% Tween 20. The second antibody, a goat anti-mouse Ig conjugated tohorseradish peroxidase (obtained from Boehringer Mannheim), was added tothe wells as 100 μl of a {fraction (1/3000)} dilution in SpecimenDiluent and incubated for one hr at RT. Plates were again washed fourtimes in PBS, Tween 20. To develop the chromogen, 100 μl of Slow TMB(3,3′,5,5′-tetramethyl benzidine obtained from Pierce Chemicals) wasadded to each well and incubated for 15 min at RT. The reaction wasstopped by the addition of 25 μl of 2 M H₂SO₄. The color intensity wasthen read on a Molecular Devices Vmax at (450 nm-650 nm).

[0351] Titers were defined as the reciprocal of the dilution of serumgiving one half the maximum OD. Maximal OD was generally taken from aninitial {fraction (1/100)} dilution, except in cases with very hightiters, in which case a higher initial dilution was necessary toestablish the maximal OD. If the 50% point fell between two dilutions, alinear extrapolation was made to calculate the final titer. To calculategeometric mean antibody titers, titers less than 100 were arbitrarilyassigned a titer value of 25.

[0352] C. Brain Tissue Preparation

[0353] After euthanasia, the brains were removed and one hemisphere wasprepared for immunohistochemical analysis, while three brain regions(hippocampus, cortex and cerebellum) were dissected from the otherhemisphere and used to measure the concentration of various Aβ proteinsand APP forms using specific ELISAs (Johnson-Wood et al., supra).

[0354] Tissues destined for ELISAs were homogenized in 10 volumes ofice-cold guanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0).The homogenates were mixed by gentle agitation using an Adams Nutator(Fisher) for three to foul hr at RT, then stored at −20° C. prior toquantitation of Aβ and APP. Previous experiments had shown that theanalytes were stable under this storage condition, and that synthetic Aβprotein (Bachem) could be quantitatively recovered when spiked intohomogenates of control brain tissue from mouse littermates (Johnson-Woodet al., supra).

[0355] D. Measurement of Aβ Levels

[0356] The brain homogenates were diluted 1:10 with ice cold CaseinDiluent (0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mMEDTA pH 8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000×g for 20min at 4° C. The synthetic Aβ protein standards (1-42 amino acids) andthe APP standards were prepared to include 0.5 M guanidine and 0.1%bovine serum albumin (BSA) in the final composition. The “total” Aβsandwich ELISA utilizes monoclonal antibody monoclonal antibody 266,specific for amino acids 13-28 of Aβ (Seubert et al., supra), as thecapture antibody, and biotinylated monoclonal antibody 3D6, specific foramino acids 1-5 of Aβ (Johnson-Wood et al., supra), as the reporterantibody. The 3D6 monoclonal antibody does not recognize secreted APP orfull-length APP, but detects only Aβ species with an amino-terminalaspartic acid. The cell line producing the antibody 3D6 has the ATCCaccession number PTA-5130, having been deposited on Apr. 8, 2003. Thisassay has a lower limit of sensitivity of ˜50 ng/ml (11 nM) and shows nocross-reactivity to the endogenous murine Aβ protein at concentrationsup to 1 ng/ml (Johnson-Wood et al., supra).

[0357] The Aβ1-42 specific sandwich ELISA employs mAβ 21F12, specificfor amino acids 33-42 of Aβ (Johnson-Wood, et al. supra), as the captureantibody. Biotinylated mAβ 3D6 is also the reporter antibody in thisassay which has a lower limit of sensitivity of about 125 μg/ml (28 μM,Johnson-Wood et al., supra). For the Aβ ELISAs, 100 μl of either mAβ 266(at 10 μg/ml) or mAβ 21F12 at (5 μg/ml) was coated into the wells of96-well immunoassay plates (Costar) by overnight incubation at RT. Thesolution was removed by aspiration and the wells were blocked by theaddition of 200 μl of 0.25% human serum albumin in PBS buffer for atleast 1 hr at RT. Blocking solution was removed and the plates werestored desiccated at 4° C. until used. The plates were rehydrated withWash Buffer [Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH7.5), plus 0.05% Tween 20] prior to use. The samples and standards wereadded in triplicate aliquots of 100 μl per well and then incubatedovernight at 4° C. The plates were washed at least three times with WashBuffer between each step of the assay. The biotinylated mAβ 3D6, dilutedto 0.5 μg/ml in Casein Assay Buffer (0.25% casein, PBS, 0.05% Tween 20,pH 7.4), was added and incubated in the wells for 1 hr at RT. Anavidin-horseradish peroxidase conjugate, (Avidin-HRP obtained fromVector, Burlingame, Calif.), diluted 1:4000 in Casein Assay Buffer, wasadded to the wells for 1 hr at RT. The calorimetric substrate, SlowTMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT,after which the enzymatic reaction was stopped by the addition of 25 μl2 N H2SO₄. The reaction product was quantified using a Molecular DevicesVmax measuring the difference in absorbance at 450 nm and 650 nm.

[0358] E. Measurement of APP Levels

[0359] Two different APP assays were utilized. The first, designatedAPP-α/FL, recognizes both APP-alpha (α) and full-length (FL) forms ofAPP. The second assay is specific for APP-α. The APP-α/FL assayrecognizes secreted APP including the first 12 amino acids of Aβ. Sincethe reporter antibody (2H3) is not specific to the α-clip-site,occurring between amino acids 612-613 of APP⁶⁹⁵ (Esch et al., Science248:1122-1124 (1990)); this assay also recognizes full length APP(APP-FL). Preliminary experiments using immobilized APP antibodies tothe cytoplasmic tail of APP-FL to deplete brain homogenates of APP-FLsuggest that approximately 30-40% of the APP-α/FL APP is FL (data notshown). The capture antibody for both the APP-α/FL and APP-α assays ismAb 8E5, raised against amino acids 444 to 592 of the APP⁶⁹⁵ form (Gameset al., supra). The reporter mAb for the APP-α/FL assay is mAb 2H3,specific for amino acids 597-608 of APP⁶⁹⁵ (Johnson-Wood et al., supra)and the reporter antibody for the APP-α assay is a biotinylatedderivative of mAb 16H9, raised to amino acids 605 to 611 of APP. Thelower limit of sensitivity of the APP-αFL assay is about 11 ng/ml (150ρM) (Johnson-Wood et al.) and that of the APP-α specific assay is 22ng/ml (0.3 nM). For both APP assays, mAb 8E5 was coated onto the wellsof 96-well EIA plates as described above for mAb 266. Purified,recombinant secreted APP-α was used as the reference standard for theAPP-α assay and the APP-α/FL assay (Esch et al., supra). The brainhomogenate samples in 5 M guanidine were diluted 1:10 in ELISA SpecimenDiluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin,0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 inSpecimen Diluent containing 0.5 M guanidine. Diluted homogenates werethen centrifuged at 16,000×g for 15 seconds at RT. The APP standards andsamples were added to the plate in duplicate aliquots and incubated for1.5 hr at RT. The biotinylated reporter antibody 2H3 or 16H9 wasincubated with samples for 1 hr at RT. Streptavidin-alkaline phosphatase(Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubatedin the wells for 1 hr at RT. The fluorescent substrate4-methyl-umbellipheryl-phosphate was added for a 30-min RT incubationand the plates were read on a Cytofluor tm 2350 fluorimeter (Millipore)at 365 nm excitation and 450 nm emission.

[0360] F. Immunohistochemistry

[0361] Brains were fixed for three days at 40C in 4% paraformaldehyde inPBS and then stored from one to seven days at 4° C. in 1%paraformaldehyde, PBS until sectioned. Forty-micron-thick coronalsections were cut on a vibratome at RT and stored in cryoprotectant (30%glycerol, 30% ethylene glycol in phosphate buffer) at −20° C. prior toimmunohistochemical processing. For each brain, six sections at thelevel of the dorsal hippocampus, each separated by consecutive 240 μmintervals, were incubated overnight with one of the followingantibodies: (1) a biotinylated anti-Aβ (mAb, 3D6, specific for human Aβ)diluted to a concentration of 2 μg/ml in PBS and 1% horse serum; or (2)a biotinylated mAb specific for human APP, 8E5, diluted to aconcentration of 3 μg/ml in PBS and 1.0% horse serum; or (3) a mAbspecific for glial fibrillary acidic protein (GFAP; Sigma Chemical Co.)diluted 1:500 with 0.25% Triton X-100 and 1/% horse serum, inTris-buffered saline, pH 7.4 (TBS); or (4) a mAb specific for CD11b,MAC-1 antigen, (Chemicon International) diluted 1:100 with 0.25% TritonX-100 and 1% rabbit serum in TBS; or (5) a mAb specific for MHC IIantigen, (Pharmingen) diluted 1:100 with 0.25% Triton X-100 and 1%rabbit serum in TBS; or (6) a rat mAb specific for CD 43 (Pharmingen)diluted 1:100 with 1% rabbit serum in PBS or (7) a rat mAb specific forCD 45RA (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (8) arat monoclonal Aβ specific for CD 45RB (Pharmingen) diluted 1:00 with 1%rabbit serum in PBS; or (9) a rat monoclonal Aβ specific for CD 45(Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (10) abiotinylated polyclonal hamster Aβ specific for CD3e (Pharmingen)diluted 1:100 with 1% rabbit serum in PBS or (11) a rat mAb specific forCD3 (Serotec) diluted 1:200 with 1% rabbit serum in PBS; or with (12) asolution of PBS lacking a primary antibody containing 1% normal horseserum.

[0362] Sections reacted with antibody solutions listed in 1,2 and 6-12above were pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide inPBS for 20 min at RT to block endogenous peroxidase. They were nextincubated overnight at 4° C. with primary antibody. Sections reactedwith 3D6 or 8E5 or CD3e mAbs were then reacted for one hr at RT with ahorseradish peroxidase-avidin-biotin-complex with kit components “A” and“B” diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,Burlingame, Calif.). Sections reacted with antibodies specific for CD45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primaryantibody were incubated for 1 hour at RT with biotinylated anti-rat IgG(Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector)diluted 1:75 in PBS, respectively. Sections were then reacted for one hrat RT with a horseradish peroxidase-avidin-biotin-complex with kitcomponents “A” and “B” diluted 1:75 in PBS (Vector Elite Standard Kit,Vector Labs, Burlingame, Calif.).

[0363] Sections were developed in 0.01% hydrogen peroxide, 0.05%3,3′-diaminobenzidine (DAB) at RT. Sections destined for incubation withthe GFAP-, MAC-1-AND MHC II-specific antibodies were pretreated with0.6% hydrogen peroxide at RT to block endogenous peroxidase thenincubated overnight with the primary antibody at 4° C. Sections reactedwith the GFAP antibody were incubated for 1 hr at RT with biotinylatedanti-mouse IgG made in horse (Vector Laboratories; Vectastain Elite ABCKit) diluted 1:200 with TBS. The sections were next reacted for one hrwith an avidin-biotin-peroxidase complex (Vector Laboratories;Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubatedwith the MAC-1-or MHC II-specific monoclonal antibody as the primaryantibody were subsequently reacted for 1 hr at RT with biotinylatedanti-rat IgG made in rabbit diluted 1:200 with TBS, followed byincubation for one hr with avidin-biotin-peroxidase complex diluted1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHCII-specific antibodies were then visualized by treatment at RT with0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for 4 and11 min, respectively.

[0364] Immunolabeled sections were mounted on glass slides (VWR,Superfrost slides), air dried overnight, dipped in Propar (Anatech) andoverlaid with coverslips using Permount (Fisher) as the mounting medium.

[0365] To counterstain Aβ plaques, a subset of the GFAP-positivesections were mounted on Superfrost slides and incubated in aqueous 1%Thioflavin S (Sigma) for 7 min following immunohistochemical processing.Sections were then dehydrated and cleared in Propar, then overlaid withcoverslips mounted with Permount.

[0366] G. Image Analysis

[0367] A Videometric 150 Image Analysis System (Oncor, Inc.,Gaithersburg, Md.) linked to a Nikon Microphot-FX microscope through aCCD video camera and a Sony Trinitron monitor was used forquantification of the immunoreactive slides. The image of the sectionwas stored in a video buffer and a color-and saturation-based thresholdwas determined to select and calculate the total pixel area occupied bythe immunolabeled structures. For each section, the hippocampus wasmanually outlined and the total pixel area occupied by the hippocampuswas calculated. The percent amyloid burden was measured as: (thefraction of the hippocampal area containing Aβ deposits immunoreactivewith mAb 3D6)×100. Similarly, the percent neuritic burden was measuredas: (the fraction of the hippocampal area containing dystrophic neuritesreactive with monoclonal antibody 8E5)×100. The C-Imaging System(Compix, Inc., Cranberry Township, Pa.) operating the Simple 32 SoftwareApplication program was linked to a Nikon Microphot-FX microscopethrough an Optronics camera and used to quantitate the percentage of theretrospenial cortex occupied by GFAP-positive astrocytes and MAC-1-andMHC II-positive microglia. The image of the immunoreacted section wasstored in a video buffer and a monochrome-based threshold was determinedto select and calculate the total pixel area occupied by immunolabeledcells. For each section, the retrosplenial cortex (RSC) was manuallyoutlined and the total pixel area occupied by the RSC was calculated.The percent astrocytosis was defined as: (the fraction of RSC occupiedby GFAP-reactive astrocytes)×100. Similarly, percent microgliosis wasdefined as: (the fraction of the RSC occupied by MAC-1-or MHCII-reactive microglia)×100. For all image analyses, six sections at thelevel of the dorsal hippocampus, each separated by consecutive 240 μmintervals, were quantitated for each animal. In all cases, the treatmentstatus of the animals was unknown to the observer.

[0368] Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein, as well as textappearing in the figures and sequence listing, are hereby incorporatedby reference in their entirety for all purposes to the same extent as ifeach were so individually denoted.

[0369] From the foregoing it will be apparent that the inventionprovides for a number of uses. For example, the invention provides forthe use of any of the antibodies to Aβ described above in the treatment,prophylaxis or diagnosis of amyloidogenic disease, or in the manufactureof a medicament or diagnostic composition for use in the same.

1 63 1 396 DNA Mus musculus CDS (1)...(396) sig_peptide (1)...(60) 1 atgatg agt cct gcc cag ttc ctg ttt ctg tta gtg ctc tgg att cgg 48 Met MetSer Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg -20 -15 -10 -5gaa acc aac ggt tat gtt gtg atg acc cag act cca ctc act ttg tcg 96 GluThr Asn Gly Tyr Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser 1 5 10 gttacc att gga caa cca gcc tcc atc tct tgc aag tca agt cag agc 144 Val ThrIle Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser 15 20 25 ctc ttagat agt gat gga aag aca tat ttg aat tgg ttg tta cag agg 192 Leu Leu AspSer Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg 30 35 40 cca ggc cagtct cca aag cgc cta atc tat ctg gtg tct aaa ctg gac 240 Pro Gly Gln SerPro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp 45 50 55 60 tct gga gtccct gac agg ttc act ggc agt gga tca ggg aca gat ttt 288 Ser Gly Val ProAsp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 aca ctg aaa atcagc aga ata gag gct gag gat ttg gga ctt tat tat 336 Thr Leu Lys Ile SerArg Ile Glu Ala Glu Asp Leu Gly Leu Tyr Tyr 80 85 90 tgc tgg caa ggt acacat ttt cct cgg acg ttc ggt gga ggc acc aag 384 Cys Trp Gln Gly Thr HisPhe Pro Arg Thr Phe Gly Gly Gly Thr Lys 95 100 105 ctg gaa atc aaa 396Leu Glu Ile Lys 110 2 132 PRT Mus musculus SIGNAL (1)...(20) 2 Met MetSer Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg -20 -15 -10 -5Glu Thr Asn Gly Tyr Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser 1 5 10Val Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser 15 20 25Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg 30 35 40Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp 45 50 5560 Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 65 7075 Thr Leu Lys Ile Ser Arg Ile Glu Ala Glu Asp Leu Gly Leu Tyr Tyr 80 8590 Cys Trp Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys 95100 105 Leu Glu Ile Lys 110 3 414 DNA Mus musculus CDS (1)...(414)sig_peptide (1)...(57) 3 atg aac ttc ggg ctc agc ttg att ttc ctt gtc cttgtt tta aaa ggt 48 Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu ValLeu Lys Gly -15 -10 -5 gtc cag tgt gaa gtg aag ctg gtg gag tct ggg ggaggc tta gtg aag 96 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly GlyLeu Val Lys 1 5 10 cct gga gcg tct ctg aaa ctc tcc tgt gca gcc tct ggattc act ttc 144 Pro Gly Ala Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly PheThr Phe 15 20 25 agt aac tat ggc atg tct tgg gtt cgc cag aat tca gac aagagg ctg 192 Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Asn Ser Asp Lys ArgLeu 30 35 40 45 gag tgg gtt gca tcc att agg agt ggt ggt ggt aga acc tactat tca 240 Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr TyrSer 50 55 60 gac aat gta aag ggc cga ttc acc atc tcc aga gag aat gcc aagaac 288 Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn65 70 75 acc ctg tac ctg caa atg agt agt ctg aag tct gag gac acg gcc ttg336 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu 8085 90 tat tat tgt gtc aga tat gat cac tat agt ggt agc tcc gac tac tgg384 Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp 95100 105 ggc cag ggc acc act gtc aca gtc tcc tca 414 Gly Gln Gly Thr ThrVal Thr Val Ser Ser 110 115 4 138 PRT Mus musculus SIGNAL (1)...(19) 4Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly -15 -10-5 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys 1 510 Pro Gly Ala Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 2025 Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Asn Ser Asp Lys Arg Leu 30 3540 45 Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser 5055 60 Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn 6570 75 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu 8085 90 Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp 95100 105 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 5 132 PRTArtificial Sequence SIGNAL (1)...(20) humanized 3D6 light chain variableregion 5 Met Met Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg-20 -15 -10 -5 Glu Thr Asn Gly Tyr Val Val Met Thr Gln Ser Pro Leu SerLeu Pro 1 5 10 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser SerGln Ser 15 20 25 Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu LeuGln Lys 30 35 40 Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser LysLeu Asp 45 50 55 60 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe 65 70 75 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val GlyVal Tyr Tyr 80 85 90 Cys Trp Gln Gly Thr His Phe Pro Arg Thr Phe Gly GlnGly Thr Lys 95 100 105 Val Glu Ile Lys 110 6 125 PRT Homo sapiens SIGNAL(1)...(13) 6 Met Gly Leu Leu Met Leu Trp Val Ser Gly Ser Ser Gly Asp IleVal -10 -5 1 Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu ProAla 5 10 15 Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn GlyTyr 20 25 30 35 Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser ProGln Leu 40 45 50 Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro AspArg Phe 55 60 65 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile SerArg Val 70 75 80 Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala LeuGln Thr 85 90 95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100105 110 7 100 PRT Homo sapiens 7 Asp Ile Val Met Thr Gln Ser Pro Leu SerLeu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg SerSer Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp TyrLeu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly SerAsn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp ValGly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro 100 8 138 PRTArtificial Sequence Humanized 3D6 heavy chain variable region 8 Met AsnPhe Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly -15 -10 -5 ValGln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 ProGly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25 SerAsn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 30 35 40 45Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser 50 55 60Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu 80 85 90Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp 95 100105 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 9 121 PRT Homosapiens 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerSer Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala AspSer Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn SerLeu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala LeuTyr Tyr Cys 85 90 95 Ala Lys Asp Asn Tyr Asp Phe Trp Ser Gly Thr Phe AspTyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 1098 PRT Homo sapiens 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheThr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly LysGly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr TyrTyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn SerLys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu AspThr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys 11 132 PRT Artificial SequenceSIGNAL (1)...(20) humanized 3D6 light chain variable region 11 Met MetSer Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg -20 -15 -10 -5Glu Thr Asn Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro 1 5 10Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser 15 20 25Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys 30 35 40Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp 45 50 5560 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 7075 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 80 8590 Cys Trp Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys 95100 105 Val Glu Ile Lys 110 12 138 PRT Artificial Sequence Humanized 3D6light chain variable region 12 Met Asn Phe Gly Leu Ser Leu Ile Phe LeuVal Leu Val Leu Lys Gly -15 -10 -5 Val Gln Cys Glu Val Gln Leu Leu GluSer Gly Gly Gly Leu Val Gln 1 5 10 Pro Gly Gly Ser Leu Arg Leu Ser CysAla Ala Ser Gly Phe Thr Phe 15 20 25 Ser Asn Tyr Gly Met Ser Trp Val ArgGln Ala Pro Gly Lys Gly Leu 30 35 40 45 Glu Trp Val Ala Ser Ile Arg SerGly Gly Gly Arg Thr Tyr Tyr Ser 50 55 60 Asp Asn Val Lys Gly Arg Phe ThrIle Ser Arg Asp Asn Ser Lys Asn 65 70 75 Thr Leu Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Val Arg Tyr Asp HisTyr Ser Gly Ser Ser Asp Tyr Trp 95 100 105 Gly Gln Gly Thr Leu Val ThrVal Ser Ser 110 115 13 393 DNA Mus musculus CDS (1)...(393) sig_peptide(1)...(57) 13 atg aag ttg cct gtt agg ctg ttg gta ctg atg ttc tgg attcct gct 48 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile ProAla -15 -10 -5 tcc agc agt gat gtt ttg atg acc caa act cca ctc tcc ctgcct gtc 96 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu ProVal 1 5 10 agt ctt gga gat caa gcc tcc atc tct tgc aga tct agt cag aacatt 144 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile15 20 25 ata cat agt aat gga aac acc tat tta gaa tgg tac ctg cag aaa cca192 Ile His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 3035 40 45 ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct240 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 5055 60 ggg gtc cca gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca288 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 6570 75 ctc aag atc aag aaa gtg gag gct gag gat ctg gga att tat tac tgc336 Leu Lys Ile Lys Lys Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys 8085 90 ttt caa ggt tca cat gtt ccg ctc acg ttc ggt gct ggg acc aag ctg384 Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 95100 105 gag ctg gaa 393 Glu Leu Glu 110 14 131 PRT Mus musculus SIGNAL(1)...(19) 14 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp IlePro Ala -15 -10 -5 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu SerLeu Pro Val 1 5 10 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser SerGln Asn Ile 15 20 25 Ile His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr LeuGln Lys Pro 30 35 40 45 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val SerAsn Arg Phe Ser 50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr 65 70 75 Leu Lys Ile Lys Lys Val Glu Ala Glu Asp Leu GlyIle Tyr Tyr Cys 80 85 90 Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly AlaGly Thr Lys Leu 95 100 105 Glu Leu Glu 110 15 426 DNA Mus musculus CDS(1)...(426) sig_peptide (1)...(57) 15 atg gac agg ctt act tcc tca ttcctg ctg ctg att gtc cct gca tat 48 Met Asp Arg Leu Thr Ser Ser Phe LeuLeu Leu Ile Val Pro Ala Tyr -15 -10 -5 gtc ctg tcc cag gct act ctg aaagag tct ggc cct gga ata ttg cag 96 Val Leu Ser Gln Ala Thr Leu Lys GluSer Gly Pro Gly Ile Leu Gln 1 5 10 tcc tcc cag acc ctc agt ctg act tgttct ttc tct ggg ttt tca ctg 144 Ser Ser Gln Thr Leu Ser Leu Thr Cys SerPhe Ser Gly Phe Ser Leu 15 20 25 agc act tct ggt atg gga gtg agc tgg attcgt cag cct tca gga aag 192 Ser Thr Ser Gly Met Gly Val Ser Trp Ile ArgGln Pro Ser Gly Lys 30 35 40 45 ggt ctg gag tgg ctg gca cac att tac tgggat gat gac aag cgc tat 240 Gly Leu Glu Trp Leu Ala His Ile Tyr Trp AspAsp Asp Lys Arg Tyr 50 55 60 aac cca tcc ctg aag agc cgg ctc aca atc tccaag gat acc tcc aga 288 Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser LysAsp Thr Ser Arg 65 70 75 aag cag gta ttc ctc aag atc acc agt gtg gac cctgca gat act gcc 336 Lys Gln Val Phe Leu Lys Ile Thr Ser Val Asp Pro AlaAsp Thr Ala 80 85 90 aca tac tac tgt gtt cga agg ccc att act ccg gta ctagtc gat gct 384 Thr Tyr Tyr Cys Val Arg Arg Pro Ile Thr Pro Val Leu ValAsp Ala 95 100 105 atg gac tac tgg ggt caa gga acc tca gtc acc gtc tcctca 426 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 110 115120 16 142 PRT Mus musculus SIGNAL (1)...(19) 16 Met Asp Arg Leu Thr SerSer Phe Leu Leu Leu Ile Val Pro Ala Tyr -15 -10 -5 Val Leu Ser Gln AlaThr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln 1 5 10 Ser Ser Gln Thr LeuSer Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu 15 20 25 Ser Thr Ser Gly MetGly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys 30 35 40 45 Gly Leu Glu TrpLeu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr 50 55 60 Asn Pro Ser LeuLys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg 65 70 75 Lys Gln Val PheLeu Lys Ile Thr Ser Val Asp Pro Ala Asp Thr Ala 80 85 90 Thr Tyr Tyr CysVal Arg Arg Pro Ile Thr Pro Val Leu Val Asp Ala 95 100 105 Met Asp TyrTrp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 110 115 120 17 136 DNAArtificial Sequence primer 17 tccgcaagct tgccgccacc atggacatgcgcgtgcccgc ccagctgctg ggcctgctga 60 tgctgtgggt gtccggctcc tccggctacgtggtgatgac ccagtccccc ctgtccctgc 120 ccgtgacccc cggcga 136 18 131 DNAArtificial Sequence primer 18 ctggggggac tggccgggct tctgcagcagccagttcagg taggtcttgc cgtcggagtc 60 cagcagggac tgggaggact tgcaggagatggaggcgggc tcgccggggg tcacgggcag 120 ggacaggggg g 131 19 146 DNAArtificial Sequence primer 19 acctgaactg gctgctgcag aagcccggccagtcccccca gcgcctgatc tacctggtgt 60 ccaagctgga ctccggcgtg cccgaccgcttctccggctc cggctccggc accgacttca 120 ccctgaagat ctcccgcgtg gaggcc 146 20142 DNA Artificial Sequence primer 20 aattctagga tccactcacg cttgatctccaccttggtgc cctggccgaa ggtgcggggg 60 aagtgggtgc cctgccagca gtagtacacgcccacgtcct cggcctccac gcgggagatc 120 ttcagggtga agtcggtgcc gg 142 21 16DNA Artificial Sequence primer 21 ctggggggac tggccg 16 22 22 DNAArtificial Sequence primer 22 acctgaactg gctgctgcag aa 22 23 138 DNAArtificial Sequence primer 23 acagaaagct tgccgccacc atggagtttgggctgagctg gctttttctt gtggctattt 60 taaaaggtgt ccagtgtgag gtgcagctgctggagtccgg cggcggcctg gtgcagcccg 120 gcggctccct gcgcctgt 138 24 135 DNAArtificial Sequence primer 24 gccgccggag cggatggagg ccacccactccaggcccttg ccgggggcct ggcgcaccca 60 ggacatgccg tagttggaga aggtgaagccggaggcggcg caggacaggc gcagggagcc 120 gccgggctgc accag 135 25 142 DNAArtificial Sequence primer 25 ctggagtggg tggcctccat ccgctccggcggcggccgca cctactactc cgacaacgtg 60 aagggccgct tcaccatctc ccgcgacaacgccaagaact ccctgtacct gcagatgaac 120 tccctgcgcg ccgaggacac cg 142 26 144DNA Artificial Sequence primer 26 ctgcaaggat ccactcaccg gaggacacggtcaccagggt gccctggccc cagtagtcgg 60 aggagccgga gtagtggtcg tagcgcacgcagtagtacag ggcggtgtcc tcggcgcgca 120 gggagttcat ctgcaggtac aggg 144 2716 DNA Artificial Sequence primer 27 gccgccggag cggatg 16 28 20 DNAArtificial Sequence primer 28 ctggagtggg tggcctccat 20 29 19 DNAArtificial Sequence primer 29 tccgcaagct tgccgccac 19 30 29 DNAArtificial Sequence primer 30 aattctagga tccactcacg cttgatctc 29 31 23DNA Artificial Sequence primer 31 acagaaagct tgccgccacc atg 23 32 22 DNAArtificial Sequence primer 32 ctgcaaggat ccactcaccg ga 22 33 10 PRTArtificial Sequence native ABeta peptide 33 Asp Ala Glu Phe Arg His AspSer Gly Tyr 1 5 10 34 402 DNA Artificial Sequence h3D6 version 1 VL 34atggacatgc gcgtgcccgc ccagctgctg ggcctgctga tgctgtgggt gtccggctcc 60tccggctacg tggtgatgac ccagtccccc ctgtccctgc ccgtgacccc cggcgagccc 120gcctccatct cctgcaagtc ctcccagtcc ctgctggact ccgacggcaa gacctacctg 180aactggctgc tgcagaagcc cggccagtcc ccccagcgcc tgatctacct ggtgtccaag 240ctggactccg gcgtgcccga ccgcttctcc ggctccggct ccggcaccga cttcaccctg 300aagatctccc gcgtggaggc cgaggacgtg ggcgtgtact actgctggca gggcacccac 360ttcccccgca ccttcggcca gggcaccaag gtggagatca ag 402 35 402 DNA ArtificialSequence h3D6 version 2 VL 35 atggacatgc gcgtgcccgc ccagctgctgggcctgctga tgctgtgggt gtccggctcc 60 tccggcgacg tggtgatgac ccagtcccccctgtccctgc ccgtgacccc cggcgagccc 120 gcctccatct cctgcaagtc ctcccagtccctgctggact ccgacggcaa gacctacctg 180 aactggctgc tgcagaagcc cggccagtccccccagcgcc tgatctacct ggtgtccaag 240 ctggactccg gcgtgcccga ccgcttctccggctccggct ccggcaccga cttcaccctg 300 aagatctccc gcgtggaggc cgaggacgtgggcgtgtact actgctggca gggcacccac 360 ttcccccgca ccttcggcca gggcaccaaggtggagatca ag 402 36 414 DNA Artificial Sequence h3D6 version 1 VH 36atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgtgag 60gtgcagctgc tggagtccgg cggcggcctg gtgcagcccg gcggctccct gcgcctgtcc 120tgcgccgcct ccggcttcac cttctccaac tacggcatgt cctgggtgcg ccaggccccc 180ggcaagggcc tggagtgggt ggcctccatc cgctccggcg gcggccgcac ctactactcc 240gacaacgtga agggccgctt caccatctcc cgcgacaacg ccaagaactc cctgtacctg 300cagatgaact ccctgcgcgc cgaggacacc gccctgtact actgcgtgcg ctacgaccac 360tactccggct cctccgacta ctggggccag ggcaccctgg tgaccgtgtc ctcc 414 37 414DNA Artificial Sequence h3D6 version 2 VH 37 atggagtttg ggctgagctggctttttctt gtggctattt taaaaggtgt ccagtgtgag 60 gtgcagctgc tggagtccggcggcggcctg gtgcagcccg gcggctccct gcgcctgtcc 120 tgcgccgcct ccggcttcaccttctccaac tacggcatgt cctgggtgcg ccaggccccc 180 ggcaagggcc tggagtgggtggcctccatc cgctccggcg gcggccgcac ctactactcc 240 gacaacgtga agggccgcttcaccatctcc cgcgacaact ccaagaacac cctgtacctg 300 cagatgaact ccctgcgcgccgaggacacc gccgtgtact actgcgtgcg ctacgaccac 360 tactccggct cctccgactactggggccag ggcaccctgg tgaccgtgtc ctcc 414 38 770 PRT Homo sapiens 38 MetLeu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 7580 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 9095 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp ValCys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr CysSer Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu LeuPro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys CysPro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala GluGlu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp TyrAla Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu GluGlu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp GluAsp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu ProTyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr ThrThr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330335 Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340345 350 Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala355 360 365 Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro GlyAsp 370 375 380 Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg LeuGlu Ala 385 390 395 400 Lys His Arg Glu Arg Met Ser Gln Val Met Arg GluTrp Glu Glu Ala 405 410 415 Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala AspLys Lys Ala Val Ile 420 425 430 Gln His Phe Gln Glu Lys Val Glu Ser LeuGlu Gln Glu Ala Ala Asn 435 440 445 Glu Arg Gln Gln Leu Val Glu Thr HisMet Ala Arg Val Glu Ala Met 450 455 460 Leu Asn Asp Arg Arg Arg Leu AlaLeu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 Gln Ala Val Pro Pro ArgPro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495 Tyr Val Arg Ala GluGln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505 510 Glu His Val ArgMet Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525 Gln Val MetThr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540 Leu SerLeu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp 545 550 555 560Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570575 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580585 590 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro595 600 605 Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His SerPhe 610 615 620 Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val GluPro Val 625 630 635 640 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr ThrArg Pro Gly Ser 645 650 655 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile SerGlu Val Lys Met Asp 660 665 670 Ala Glu Phe Arg His Asp Ser Gly Tyr GluVal His His Gln Lys Leu 675 680 685 Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met Val Gly Gly Val Val IleAla Thr Val Ile Val Ile Thr Leu 705 710 715 720 Val Met Leu Lys Lys LysGln Tyr Thr Ser Ile His His Gly Val Val 725 730 735 Glu Val Asp Ala AlaVal Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750 Gln Gln Asn GlyTyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755 760 765 Gln Asn 77039 40 DNA Artificial Sequence primer 39 actagtcgac atgaagttgc ctgttaggctgttggtgctg 40 40 39 DNA Artificial Sequence primer 40 actagtcgacatggagwcag acacactcct gytatgggt 39 41 40 DNA Artificial Sequence primer41 actagtcgac atgagtgtgc tcactcaggt cctggsgttg 40 42 43 DNA ArtificialSequence primer 42 actagtcgac atgaggrccc ctgctcagwt tyttggmwtc ttg 43 4340 DNA Artificial Sequence primer 43 actagtcgac atggatttwc aggtgcagattwtcagcttc 40 44 37 DNA Artificial Sequence primer 44 actagtcgacatgaggtkcy ytgytsagyt yctgrgg 37 45 41 DNA Artificial Sequence primer 45actagtcgac atgggcwtca agatggagtc acakwyycwg g 41 46 41 DNA ArtificialSequence primer 46 actagtcgac atgtggggay ctktttycmm tttttcaatt g 41 4735 DNA Artificial Sequence primer 47 actagtcgac atggtrtccw casctcagttccttg 35 48 37 DNA Artificial Sequence primer 48 actagtcgac atgtatatatgtttgttgtc tatttct 37 49 38 DNA Artificial Sequence primer 49 actagtcgacatggaagccc cagctcagct tctcttcc 38 50 27 DNA Artificial Sequence primer50 ggatcccggg tggatggtgg gaagatg 27 51 37 DNA Artificial Sequence primer51 actagtcgac atgaaatgca gctgggtcat sttcttc 37 52 36 DNA ArtificialSequence primer 52 actagtcgac atgggatgga gctrtatcat sytctt 36 53 37 DNAArtificial Sequence primer 53 actagtcgac atgaagwtgt ggttaaactg ggttttt37 54 35 DNA Artificial Sequence primer 54 actagtcgac atgractttgggytcagctt grttt 35 55 40 DNA Artificial Sequence primer 55 actagtcgacatggactcca ggctcaattt agttttcctt 40 56 37 DNA Artificial Sequence primer56 actagtcgac atggctgtcy trgsgctrct cttctgc 37 57 36 DNA ArtificialSequence primer 57 actagtcgac atggratgga gckggrtctt tmtctt 36 58 33 DNAArtificial Sequence primer 58 actagtcgac atgagagtgc tgattctttt gtg 33 5940 DNA Artificial Sequence primer 59 actagtcgac atggmttggg tgtggamcttgctattcctg 40 60 37 DNA Artificial Sequence primer 60 actagtcgacatgggcagac ttacattctc attcctg 37 61 38 DNA Artificial Sequence primer 61actagtcgac atggattttg ggctgatttt ttttattg 38 62 37 DNA ArtificialSequence primer 62 actagtcgac atgatggtgt taagtcttct gtacctg 37 63 27 DNAArtificial Sequence primer 63 ggatcccggg agtggataga ctgatgg 27

We claim:
 1. Humanized 10D5 antibody.
 2. A humanized antibody, or fragment thereof, comprising a humanized light chain comprising three light chain complementarily determining regions (CDRs) from the mouse monoclonal antibody 10D5 and a light chain variable region framework sequence from a human immunoglobulin light chain; and a humanized heavy chain comprising three heavy chain CDRs from the mouse monoclonal antibody 10D5 and a heavy chain variable region framework sequence from a human immunoglobulin heavy chain; wherein the light chain CDRs have the following amino acid sequences: light chain CDR1: Arg Ser Ser Gln Asn Ile Ile His Ser Asn Gly (residues 24-39 of SEQ ID NO:14) Asn Thr Tyr Leu Glu

light chain CDR2: Lys Val Ser Asn Arg Phe Ser (residues 55-61 of SEQ ID NO:14)

light chain CDR3: Phe Gln Gly Ser His Val Pro Leu Thr (residues 94-102 of SEQ ID NO:14)

and the heavy chain CDRs have the following amino acid sequences: heavy chain CDR1: Thr Ser Gly Met Gly Val Ser (residues 31-37 of SEQ ID NO:16)

heavy chain CDR2: His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser (residues 52-67 of SEQ ID NO: 16) and,

heavy chain CDR3: Arg Pro Ile Thr Pro Val Leu Val Asp Ala Met Asp Tyr (residues 100-112 of SEQ ID NO: 16).


3. An antibody fragment obtainable by enzymatic cleavage of the humanized antibody of any one of claims 1-2.
 4. An Fab or F(ab′)2 fragment of any one of the humanized antibodies of claims 1-2.
 5. The F(ab′)2 fragment of claim
 4. 6. The Fab fragment of claim
 4. 7. The humanized antibody or fragment of any one of claims 1-6, which is a single chain antibody.
 8. The humanized antibody or fragment of any one of claims 1-7 that is an IgG1 immunoglobulin isotype.
 9. The humanized antibody or fragment of any one of claims 1-8, wherein the antibody or fragment thereof is produced in a host cell selected from the group consisting of a myeloma cell and a chinese hamster ovary (CHO) cell.
 10. A polynucleotide compound, comprising a sequence coding for the light chain or the heavy chain of the humanized antibody of any one of claims 1-9, or a fragment thereof.
 11. A polynucleotide sequence, which when expressed in a suitable host cell, yields an antibody of any one of claims 1-9.
 12. An expression vector for expressing the antibody of any one of claims 1-9 comprising the polynucleotide sequence of any one of claims 10-11.
 13. A cell transfected with the expression vector of claim
 12. 14. A cell transfected with two expression vectors of claim 12, wherein a first vector comprises the polynucleotide sequence coding for the light chain and a second vector comprises the sequence coding for the heavy chain.
 15. A cell that is capable of expressing the humanized antibody or fragment of any one of claims 1-9.
 16. The cell of any one of claims 13-15, wherein the cell is selected from the group consisting of a myeloma cell and a chinese hamster ovary (CHO) cell.
 17. A pharmaceutical composition comprising the humanized antibody or fragment of any one of claims 1-9, and a pharmaceutically acceptable excipient.
 18. A method of treating Down's syndrome or clinical or pre-clinical Alzheimer's disease in a human subject, comprising administering to the human subject an effective amount of a humanized antibody or fragment of any one of claims 1-9.
 19. A method to inhibit the formation of Aβ plaque in the brain of a human subject, comprising administering to the human subject an effective amount of the humanized antibody or fragment of any one of claims 1-9.
 20. A method to reduce Aβ plaque in the brain of a human subject, comprising administering to the human subject an effective amount of a humanized antibody or fragment of any one of claims 1-9.
 21. The method of either of claims 19-20, wherein the subject is diagnosed with clinical or pre-clinical Alzheimer's disease or Down's syndrome.
 22. The method of any one of claims 19-20, wherein the subject is not diagnosed with clinical or pre-clinical Alzheimer's disease or Down's syndrome.
 23. Use of the humanized antibody or a fragment thereof according to any one of claims 1-9 for the manufacture of a medicament, including prolonged expression of recombinant sequences of the antibody or antibody fragment in human tissues, for treating clinical or pre-clinical Alzheimer's disease or Down's syndrome.
 24. Use of the humanized antibody or fragment of any one of claims 1-9 for the manufacture of a medicament for treating Alzheimer's disease. 